参见作者原研究论文

本实验方案简略版
Feb 2020

本文章节


 

Generation and Implementation of Reporter BHK-21 Cells for Live Imaging of Flavivirus Infection
黄病毒感染活体显像报告细胞BHK-21的制备与实施   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

The genus Flavivirus within the family Flaviviridae includes many viral species of medical importance, such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV), among others. Presently, the identification of flavivirus-infected cells is based on either the immunolabeling of viral proteins, the application of recombinant reporter replicons and viral genomes, or the use of cell-based molecular reporters of the flaviviral protease NS2B-NS3 activity. Among the latter, our flavivirus-activatable GFP and mNeptune reporters contain a quenching peptide (QP) joined to the fluorescent protein by a linker consisting of a cleavage site for the flavivirus NS2B-NS3 proteases (AAQRRGRIG). When the viral protease cleaves the linker, the quenching peptide is removed, and the fluorescent protein adopts a conformation promoting fluorescence. Here we provide a detailed protocol for the generation, selection and implementation of stable BHK-21 cells expressing our flavivirus genetically-encoded molecular reporters, suitable to monitor the viral infection by live-cell imaging. We also describe the image analysis procedures and provide the required software pipelines. Our reporter cells allow the implementation of single-cell infection kinetics as well as plaque assays for both reference and native strains of flaviviruses by live-cell imaging.


Graphic abstract:



Workflow for the generation and implementation of reporter BHK-21 cells for live imaging of flavivirus infection.


Keywords: Flavivirus (黄病毒属), Fluorescence (荧光), NS2B-NS3 (NS2B-NS3), Protease (蛋白酶), Live-cell imaging (活细胞成像), Reporter cells (报告细胞), Plaque assay (噬斑试验), Image analysis (图象分析)

Background

Flaviviruses represent an emerging and re-emerging global threat that cause diseases both in animals and humans, including many medically relevant viruses like yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DENV), and Zika virus (ZIKV), among others (Gould and Solomon, 2008). At present, the detection of flavivirus-infected cells is based on either the antibody labeling of viral proteins (Balsitis et al., 2008), the use of recombinant reporter replicons and viral genomes (Li et al., 2013; Schmid et al., 2015; Xie et al., 2016; Tamura et al., 2017; Kümmerer, 2018), or the application of genetically-encoded molecular reporters of the flavivirus NS2B-NS3 proteolytic activity (Medin et al., 2015; Hsieh et al., 2017; McFadden et al., 2018). Immunolabeling implies both fixation and permeabilization which hamper their implementation for studies in living cells. Reporter replicons and viral genomes are suitable for live-cell imaging assays, but they are restricted to particular molecular clones mainly derived from reference strains and thus, not applicable when working with clinical isolates or native viral strains. In this context, cell-based molecular reporters of the flaviviral proteases constitute a favorable alternative for the study of native flavivirus strains by live-cell imaging. Based on our recently published flavivirus-activatable GFP (FlaviA-GFP) and flavivirus-activatable mNeptune (FlaviA-mNeptune) reporters (Arias-Arias et al., 2020), here we describe in detail a protocol for the generation, selection, and implementation of stably-transduced reporter BHK-21 cells for live imaging of flavivirus infection in single cells and viral plaques. Furthermore, we provide a rationale for a software-based image analysis approach to demonstrate the capabilities of this reporter cell line for single-cell and viral-plaque tracking. In addition, we include the optimized CellProfiler analysis pipelines for studies employing this or other cell-based reporters. Our approach represents the first fluorescence activatable cell-based reporter system for monitoring the kinetics of infection by both reference and native strains of flaviviruses like DENV, ZIKV, and YFV using live-cell imaging.

Materials and Reagents

  1. Cell culture flasks, 75 cm2 (Greiner Bio-One, CELLSTAR®, catalog number: 658175 )

  2. Cell culture dishes, 100/20 mm (Greiner Bio-One, CELLSTAR®, catalog number: 664160 )

  3. 2 ml reaction tubes (Greiner Bio-One, catalog number: 623201 )

  4. 3 ml sterile syringes (Ultident Scientific, catalog number: BD-309657 )

  5. Syringe filters 0.2 μm hydrophilic polyethersulfone, 32 mm diameter (Pall, Acrodisc®, catalog number: 4652 )

  6. 10 ml sterile syringes (Ultident Scientific, catalog number: BD-302995 )

  7. Syringe filters 0.45 μm hydrophilic cellulose acetate, 28 mm diameter (Sartorius, Minisart®, catalog number: 16555 )

  8. 15 ml conical tubes (Greiner Bio-One, CELLSTAR®, catalog number: 188271 )

  9. 0.5 ml reaction tubes (Greiner Bio-One, catalog number: 667201 )

  10. Hexadimethrine bromide (Merck, Sigma-Aldrich, catalog number: H9268 )

  11. 48-well cell culture plates (Greiner Bio-One, CELLSTAR®, catalog number: 677180 )

  12. 1.5 ml reaction tubes (Greiner Bio-One, catalog number: 616201 )

  13. 12-well cell culture plates (Greiner Bio-One, CELLSTAR®, catalog number: 665180 )

  14. 1.5 ml light protection reaction tubes (Greiner Bio-One, catalog number: 616283 )

  15. Cell culture flasks, 25 cm2 (Greiner Bio-One, CELLSTAR®, catalog number: 690175 )

  16. 6-well cell culture plates (Greiner Bio-One, CELLSTAR®, catalog number: 657160 )

  17. 96-well black cell culture plates (Greiner Bio-One, µCLEAR®, catalog number: 655096 )

  18. 24-well cell culture plates (Greiner Bio-One, CELLSTAR®, catalog number: 662160 )

  19. HEK 293T cells (ATCC, catalog number: CRL-3216 )

  20. Plasmids:

    pLenti-FlaviA-GFP-puro (a gift from Jorge L. Arias-Arias, Addgene plasmid #140088)

    pLenti-CMV-FlaviA-mNeptune-puro (a gift from Jorge L. Arias-Arias, Addgene plasmid #140091)

    pMD2.G (a gift from Didier Trono, Addgene plasmid #12259)

    psPAX2 (a gift from Didier Trono, Addgene plasmid # 12260 )

  21. LB agar plates with 100 µg/ml ampicillin (Merck, Sigma-Aldrich, catalog number: L5667 )

  22. LB broth (Miller) (Merck, Sigma-Aldrich, catalog number: L2542 )

  23. 100 mg/ml ampicillin solution (Merck, Sigma-Aldrich, catalog number: A5354 )

  24. NucleoSpin plasmid mini kit (Macherey-Nagel, catalog number: 740588.5 0)

  25. DMEM, high glucose, GlutaMAXTM, pyruvate (Thermo Fisher Scientific, Gibco, catalog number: 10569044 )

  26. Antibiotic-antimycotic 100× (Thermo Fisher Scientific, Gibco, catalog number: 15240062 )

  27. Fetal bovine serum (FBS) qualified, heat inactivated (Thermo Fisher Scientific, Gibco, catalog number: 10438-026 )

  28. Polyethylenimine (PEI), linear, MW 25000, transfection grade (Polysciences, PEI 25KTM, catalog number: 23966-1 )

  29. UltraPureTM DNase/RNase-free distilled water (Thermo Fisher Scientific, Invitrogen, catalog number: 10977015 )

  30. Hydrochloric acid, 36.5-38.0%, BioReagent (Merck, Sigma-Aldrich, catalog number: H1758 )

  31. BHK-21 [C-13] (ATCC, catalog number: CCL-10 )

  32. MEM, GlutaMAXTM supplement (Thermo Fisher Scientific, Gibco, catalog number: 41090101 ).

  33. Sodium pyruvate, 100 mM (Thermo Fisher Scientific, Gibco, catalog number: 11360070 ).

  34. PBS, pH 7.4 (Thermo Fisher Scientific, Gibco, catalog number: 10010023 ).

  35. TrypLETM express enzyme (1×), no phenol red (Thermo Fisher Scientific, Gibco, catalog number: 12604013 )

  36. Puromycin dihydrochloride from Streptomyces alboniger (Merck, Sigma-Aldrich, catalog number: P8833 )

  37. Clinical isolate DENV-2/CR/13538/2007 (Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud, Cartago, Costa Rica) (Soto-Garita et al., 2016)

  38. Vaccine strain YFV/US/17D/1937 (Sanofi Pasteur, YF-VAX®)

  39. FluoroBriteTM DMEM (Thermo Fisher Scientific, Gibco, catalog number: A1896701 )

  40. GlutaMAXTM supplement (Thermo Fisher Scientific, Gibco, catalog number: 35050061 )

  41. Minimum essential medium eagle AutoModTM (Merck, Sigma-Aldrich, catalog number: M0769 )

  42. Carboxymethylcellulose sodium salt (Merck, Sigma-Aldrich, catalog number: C4888 )

  43. Sodium bicarbonate (Merck, Sigma-Aldrich, catalog number: S5761 )

  44. Complete DMEM (see Recipes)

  45. PEI solution (1 mg/ml) (see Recipes)

  46. Polybrene solution (10 mg/ml) (see Recipes)

  47. Complete MEM (see Recipes)

  48. PBS 1% FBS (see Recipes)

  49. Puromycin solution (10 mg/ml) (see Recipes)

  50. FluoroBriteTM DMEM 2% FBS (see Recipes)

  51. Plaque media 2% FBS (see Recipes)

Equipment

  1. Biological safety cabinet (ESCO, Labculture® Class II, Type A2, catalog number: LA2-3A2-E )

  2. CO2 incubator (Thermo Scientific, model: Forma Series II, catalog number: 3110 )

  3. 4 °C refrigerator (Thermo Scientific, Value Lab, catalog number: 20LREETSA )

  4. -20 °C freezer (Thermo Scientific, Value Lab, catalog number: 20LFEETSA )

  5. -80 °C freezer (Sanyo, VIP series, catalog number: MDF-U32V )

  6. Water bath (PolyScience, catalog number: WBE20A12E )

  7. Centrifuge (Eppendorf, model: 5810 )

  8. Microcentrifuge (Eppendorf, model: 5418 )

  9. Pipettes (Thermo Scientific, FinnpipetteTM F2 GLP Kit, catalog number: 4700880 )

  10. Pipet filler (Thermo Scientific, S1, catalog number: 9511 )

  11. Hemocytometer (Boeco, Neubauer improved, catalog number: BOE 13 )

  12. Shaking incubator (Shel Lab, catalog number: SSI3 )

  13. ThermoMixer® C block (Eppendorf, catalog number: 5382000015 )

  14. Vortex mixer (Thermo Scientific, MaxiMixTM, catalog number: M16715Q )

  15. Ultraviolet crosslinker (UVP, CL-100, catalog number: UVP95017401 )

  16. NanoDropTM 2000 spectrophotometer (Thermo Scientific, catalog number: ND-2000 )

  17. Steam sterilizer (Yamato, catalog number: SQ510 )

  18. Water purification system (Merck, Milli-Q advantage A10)

  19. Inverted microscope (Nikon, Eclipse, catalog number: TS100 )

  20. Flow cytometer (BD Biosciences, BD AccuriTM C6)

  21. Cell sorter (BD Biosciences, BD FACSJazzTM)

  22. Automated fluorescence microscope (Biotek, Lionheart FX ) with:

    CO2 gas controller (Biotek, catalog number: 1210012 )

    Humidity chamber (Biotek, catalog number: 1450006 )

    4× objective (Biotek, catalog number: 1220519 )

    20× objective (Biotek, catalog number: 1220517 )

    GFP filter cube (Biotek, catalog number: 1225101 )

    465 nm LED cube (Biotek, catalog number: 1225001 )

    Cy5 filter cube (Biotek, catalog number: 1225105 )

    623 nm LED cube (Biotek, catalog number: 1225005 )

Software

  1. Gen5 Image+ (Biotek, https://www.biotek.com)

  2. CellProfiler 4.0 (Broad Institute, https://cellprofiler.org/releases)

Procedure

  1. Lentiviral vectors assembly and titration

    Assembly
    1. Prepare plasmid stocks following standard molecular biology procedures (https://www.jove.com/v/5062/plasmid-purification) and the protocol provided by the manufacturer of the NucleoSpin plasmid mini kit (Macherey-Nagel).

    2. Manually seed 6,000,000 HEK 293T cells in 100/20 mm cell culture dishes with 8 ml of DMEM 10% FBS (Recipe 1) and incubate overnight at 37 °C and 5% CO2 to reach 70-80% confluency. For a detailed procedure of seeding cells please refer to the bio-protocol paper by Freppel et al., 2018 (reference 3).

    3. Using a pipette, replace the medium by removing and discarding all the DMEM 10% FBS in the dishes (~8 ml) and adding 4 ml of fresh DMEM 2% FBS (Recipe 1).

    4. Prepare the transfection mix as follows:

      Suspension A: 45 µl PEI 1 mg/ml (Recipe 2) + 955 µl unsupplemented DMEM (without FBS and antibiotic-antimycotic).

      Suspension B: 6 µg pLenti-FlaviA-GFP-puro or pLenti-CMV-FlaviA-mNeptune-puro + 6 µg pMD2.G + 6 µg psPAX2 and bring to 1,000 µl with unsupplemented DMEM.

      Mix suspensions A with B and incubate for 15 min at room temperature.

    5. Add the 2 ml transfection mix to the cells (dropwise) and incubate overnight at 37 °C and 5% CO2.

    6. Replace the medium with 6 ml of fresh DMEM 2% FBS and incubate for 48 h at 37 °C and 5% CO2.

    7. Harvest culture supernatants and filter them (0.45-μm pore size) to eliminate cells and debris. Use filters with low protein adherence, like cellulose acetate or polyethersulfone (PES). Do not use nitrocellulose filters as they could bind the lentiviral particles.

    8. Prepare 500 µl aliquots and store at -80 °C.


    Titration
    1. As described above, seed 50,000 BHK-21 cells per well in a 48-well cell culture plate with 500 µl/well of MEM 2% FBS (Recipe 4) and incubate overnight at 37 °C and 5% CO2. Prepare extra wells to be used as control cells.

    2. Thaw in a water bath (37 °C) one of the lentiviral 500 µl stock aliquots and add polybrene (Recipe 3) at a final concentration of 5 µg/ml.

    3. Prepare 10-fold serial dilutions (10-1-10-6) of the lentiviral stock in 1.5 ml reaction tubes. Start mixing 50 µl of the lentiviral particles in 450 µl of DMEM 2% FBS + 5 µg/ml polybrene (10-1). Vortex the tube for 5 s and mix 50 µl of the 10-1 lentiviral dilution in 450 µl of DMEM 2% FBS + 5 µg/ml polybrene (10-2). Repeat this procedure for the other dilutions (10-3-10-6) and incubate for 15 min at room temperature. Change the tip between dilutions to avoid cross-contamination. For a detailed and graphical explanation of 10-fold serial dilutions preparation please refer to the bio-protocol paper by Freppel et al., 2018 (reference 3).

    4. Replace the medium of the cells with 150 µl/well of undiluted lentiviral stock and each serial dilution thereof. Add 150 µl/well of DMEM 2% FBS + 5 µg/ml polybrene to the control cells. To avoid cross contamination, make the inoculation of the samples in order, starting with the control, following with the serial dilutions (from higher to lower dilution), and ending with the undiluted lentiviral stock.

    5. Centrifuge at 300 × g for 2 h at 25 °C for viral adsorption.

    6. Replace the inoculum with 500 µl/well of DMEM 2% FBS and incubate for 48 h at 37 °C and 5% CO2.

    7. Discard the medium, wash the cells once with 100 µl/well of PBS, add 100 µl/well of TrypLETM express (no phenol red), incubate for 5 min at 37 °C, and resuspend the cells with 400 µl/well of PBS 1% FBS (Recipe 5).

    8. Based on the basal background of the reporter proteins and by comparison with the non-transduced control cells (Figure 1), determine by flow cytometry the percentage of GFP+ (488 nm laser - 530/30 nm filter) or mNeptune+ (640 nm laser -675/25 nm filter) cells present in the samples infected with different dilutions of the lentiviral seed. For a detailed procedure about flow cytometry of fluorescent proteins please refer to the protocol by Hawley et al., 2004 (reference 5).

    9. Using the data of the higher dilution with detectable transduced cells, calculate the biological titer in transducing units per milliliter (TU/ml), applying the following formula:


      TU/ml = (P × N / 100 × V) × 1/DF


      where P = % GFP+ or mNeptune+ cells, N = number of cells at transduction = 50,000, V = volume of inoculum per well = 0.15 ml, and DF = dilution factor = 1 (undiluted), 10–1 (diluted 1/10), 10–2 (diluted 1/100), and so on (Tiscornia et al., 2006).


  2. Reporter cell lines production and selection

    Production

    1. As described above, seed 100,000 BHK-21 cells per well in a 12-well cell culture plate with 1 ml/well of MEM 2% FBS and incubate overnight at 37 °C and 5% CO2. Prepare an extra well for the control cells.

    2. Thaw in a water bath (37 °C) one of the 500 µl stock aliquots of lentiviral particles carrying genetic constructs codifying for either the FlaviA-GFP or the FlaviA-mNeptune reporters and add polybrene at a final concentration of 5 µg/ml.

    3. Based on the previously calculated biological titer in TU/ml, prepare 500 µl/well of lentiviral inoculum at a multiplicity of infection (MOI) of 1 (1 TU per cell). As 100,000 cells per well were plated in manteinance medium (MEM 2% FBS), 100,000 TU must be diluted in MEM 2% FBS + 5 µg/ml polybrene to a final volume of 500 μl. Incubate for 15 min at room temperature.

    4. Replace the medium of the cells with the 500 µl/well of lentiviral inoculum. Add 500 µl/well of MEM 2% FBS + 5 µg/ml polybrene to the control cells.

    5. Centrifuge at 300 × g for 2 h at 25 °C for viral adsorption.

    6. Replace the inoculum with 1 ml/well of MEM 2% FBS and incubate for 48 h at 37 °C and 5% CO2.

    7. Based on the basal background of the reporter proteins, monitor the effectiveness of the transduction by fluorescence microscopy in the green/GFP (FlaviA-GFP) and far-red/Cy5 (FlaviA-mNeptune) channels.


    Selection

    1. For the antibiotic selection of transduced cells, replace the medium with 750 µl/well of MEM 10% FBS containing 8 µg/ml of puromycin (Recipe 6) and incubate overnight at 37 °C and 5% CO2. Apply the same treatment to the control cells.

    2. Replace the medium of both transduced and control cells with 1 ml/well of MEM 10% FBS containing 4 µg/ml of puromycin. Incubate at 37 °C and 5% CO2 until 100% mortality of the control cells is evidenced by light microscopy (commonly 24-48 h).

    3. Replace the medium of the selected cells with 1 ml/well of MEM 10% FBS + 0.5 µg/ml puromycin and incubate at 37 °C and 5% CO2 until reaching confluency of 80-90%.

    4. Passage the selected cells to 25 cm culture flasks with 5 ml of MEM 10% FBS + 0.5 µg/ml puromycin and incubate at 37 °C and 5% CO2 until reaching a confluency of 80-90% (https://www.jove.com/v/5052/passaging-cells).

    5. For the FACS selection, discard the medium, wash the cells once with 1 ml of PBS, add 500 µl of TrypLETM express (no phenol red), incubate for 5 min at 37 °C, and resuspend the cells with 1 ml of PBS 1% FBS.

    6. Count the cells with a hemocytometer (https://www.jove.com/v/5048/using-a-hemacytometer-to-count-cells) and prepare 2 ml of cell suspensions at 1,000,000 cells/ml in PBS 1% FBS.

    7. Aspirate the cell suspensions into the cell sorter. For the FlaviA-GFP reporter isolate at least two cell subpopulations with different but homogeneous levels of the reporter's basal background using the FL1 detector (488 nm laser - 530/30 nm filter) (Figure 1). Apply the same procedure to the FlaviA-mNeptune reporter but using the FL4 detector (640 nm laser - 675/25 nm filter).

      Note: The basal background of the FlaviA-GFP and FlaviA-mNeptune reporters make them sensitive to the levels of cellular expression: If the expression is too high the background will mask the signal produced upon activation of the reporters. If the expression is too low there will not be enough reporter’s signal over the background.



      Figure 1. FACS analysis of BHK-21/FlaviA-GFP stable cells. A. Histogram showing the difference in the green fluorescence (FL1 detector- 530/30 nm filter) between wild-type and reporter BHK-21 cells due to the basal background of the FlaviA-GFP reporter proteins. B. Scatter plots showing the heterogeneity in the population of stable BHK-21 cells with different levels of expression of the FlaviA-GFP reporter. For the selection of the best reporter cells (highest signal-to-noise ratio), at least two cell subpopulations with different (low, medium or high) but homogeneous levels of the reporter's basal background must be isolated and tested by live-cell imaging upon flavivirus infection.


    8. Seed the isolated cell suppopulations in different wells of a 48-well cell culture plate with 1 ml/well of MEM 10% FBS + 0.5 µg/ml puromycin and incubate at 37 °C and 5% CO2 until reaching a confluency of 80-90%.

    9. Passage the selected cells to a 6-well cell culture plate with 3 ml of MEM 10% FBS + 0.5 µg/ml puromycin and incubate at 37 °C and 5% CO2 until reaching a confluency of 80-90%.

    10. Test the different isolated cell suppopulations by a live-cell imaging flavivirus infection kinetics according to the protocol described in Procedure C.

      Note: The best reporter cells will be those with the highest signal-to-noise ratio upon flavivirus infection. The fluorescence signal-to-noise ratio is calculated by dividing the signal of the reporter cells treated with infectious virus by the noise given by the reporter cells treated with UV-inactivated virus at the same post-inoculation time.


  3. Infection kinetics in reporter cells by live-cell imaging

    1. Prepare and titer the flaviviral seed of your choice (e.g., DENV, ZIKV, or YFV) according tostandard virological methodologies (Medina et al., 2012; Freppel et al., 2018). For viral inactivation, place 200 µl/well of the flaviviral seed in a 24-well cell culture plate, remove the lid and apply 5 cycles of UV light (254 nm) exposure at an energy of 400,000 µJ/cm2 into an ultraviolet crosslinker. Between cycles, shake the plate for 5 s using your hands.

    2. As described above, seed 15,000 BHK-21/FlaviA-GFP stable cells per well in a µClear black 96-well plate with 100 µl/well of MEM 2% FBS and incubate overnight at 37 °C and 5% CO2.

    3. Based on the calculated titer of the flaviviral seed in plaque forming units (PFU)/ml, prepare 50 µl/well of inoculum at a low MOI (between 0.1 and 0.25). As 15,000 cells per well were plated in maintenance medium, between 1,500 and 3,750 PFUs must be diluted in MEM 2% FBS to a final volume of 50 μl/well. Multiply for the total number of wells to be inoculated and prepare a single inoculum suspension. Likewise, prepare the UV-inactivated inoculum suspension.

    4. Replace the medium of the cells with 50 µl/well of either the infectious or the UV-inactivated flaviviral inoculum and incubate for 2 h at 37 °C and 5% CO2 for viral adsorption. Using your hands, shake the plate for 5 s every 15 min.

    5. Replace the inoculum with 150 µl/well of FluoroBriteTM DMEM 2% FBS (Recipe 7) and incubate for the desired time of your kinetics (e.g., 120 h) at 37 °C and 5% CO2 into the automated fluorescence microscope. Add 200 µl/well of PBS to the surrounding wells to avoid desiccation.

    6. Using your microscope’s software (e.g., Gen5 Image+), program the image acquisition with the 4× or the 20× objective in the green/GFP channel at the desired post-infection times (Figure 2A).

      Note: The acquisition parameters (excitation intensity, exposure time, and camera gain) of the images are variable according to the particular reporter cell subpopulation isolated by FACS. Always include an extra well of control cells for the adjustment of the acquisition parameters as the overexposure to the excitation light may harm the cells of the experimental conditions. Our recommendation is to set those parameters to a level where the background signal of the reporter proteins is just perceptible in the first images of the kinetics, in order to increase the dynamic range of the reporter’s fluorescence upon activation by flaviviral proteases.



      Figure 2. DENV-2 infection kinetics in reporter BHK-21 cells by live-cell imaging. A. Stable BHK-21 cells expressing the FlaviA-GFP reporter were inoculated with either infectious or UV-inactivated DENV-2 13538 at a low MOI of 0.1 and captured by live-cell imaging at the specified time periods. Magnification of 40×, scale bar = 100 µm. B. The image analysis of the infection kinetics with the software CellProfiler 4.0 allowed the tracking of single cells over time based on the reported fluorescence (each colored line in the graphs corresponds to an individual cell). The black dashed lines represent the mean values of cell fluorescence.


  4. Kinetic plaque assay in reporter cells by live-cell imaging

    1. As described above, seed 25,000 BHK-21/FlaviA-mNeptune stable cells per well in a µClear black 96-well plate with 100 µl/well of MEM 10% FBS and incubate overnight at 37 °C and 5% CO2.

    2. Prepare 10-fold serial dilutions (10-1-10-6) of the flaviviral seed in 1.5 ml reaction tubes. Start mixing 50 µl of the flaviviral seed in 450 µl of MEM 2% FBS (10-1). Vortex the tube for 5 s and mix 50 µl of the 10-1 flaviviral dilution in 450 µl of MEM 2% FBS (10-2). Repeat this procedure for the other dilutions (10-3-10-6). Change the tip between dilutions to avoid cross-contamination. For a detailed and graphical explanation of 10-fold serial dilutions preparation please refer to the bio-protocol paper by Freppel et al., 2018 (reference 3).

    3. Replace the medium of the cells with 50 µl/well of each serial dilution of the flaviviral seed. Inoculate a control well with 50 µl of UV-inactivated flaviviral seed. Incubate for 2 h at 37 °C and 5% CO2 for viral adsorption. Using your hands, shake the plate for 5 s every 15 min.

    4. Replace the inoculum with 150 µl/well of plaque media 2% FBS (Recipe 8) and incubate for 120 h at 37 °C and 5% CO2 into the automated fluorescence microscope. Add 200 µl/well of PBS to the surrounding wells to avoid desiccation.

    5. Using your microscope’s software (e.g., Gen5 Image+), program the image acquisition (a montage of the whole well) with the 4× objective in the far-red/Cy5 channel at the desired post-infection times (Figure 3A).

      Note: The acquisition parameters (excitation intensity, exposure time, and camera gain) of the images are variable according to the particular reporter cell suppopulation isolated by FACS. Always include an extra well of control cells for the adjustment of the acquisition parameters, as the overexposure to the excitation light may harm the cells of the experimental conditions. Our recommendation is to set those parameters to a level where the background signal of the reporter proteins is just perceptible in the first images of the kinetics, in order to increase the dynamic range of the reporter’s fluorescence upon activation by flaviviral proteases.



      Figure 3. YFV kinetic plaque assay in reporter BHK-21 cells by live-cell imaging. A. Stable BHK-21 cells expressing the FlaviA-mNeptune reporter were inoculated with decimal dilutions (10-1-10-6) of either infectious or UV-inactivated YFV 17D. After addition of plaque media 2% FBS, entire wells of the plate were captured by live-cell imaging at the specified time periods. Magnification of 40×, scale bar = 1,000 µm. B. The image analysis of the kinetic plaque assay with the software CellProfiler 4.0 allowed the identification of single viral plaques (upper panel) and the tracking of those plaques over time (lower panel). For a deeper exemplification of the results obtained with our kinetic plaque assay please watch the live-cell imaging video (Video 1).


      Video 1. ZIKV plaque assay kinetics on reporter BHK-21 cells

Data analysis

  1. Infection kinetics in reporter cells

Single cell tracking of infected cells based on its fluorescence (Figure 2B) was performed using our CellProfiler pipeline for single cell analysis (“SingleCellsTracking.cpproj”, Figure 4). To perform this analysis just drag and drop the images in the Images module and press Analyze Images. In order to modify the pipeline for your cells and conditions, go to Start Test Mode, adjust the parameters described in the modules (one by one) and press Step to see the output of the modifications applied to a particular module. Once all the modifications are ready, press Exit Test Mode and go to Analyze Images. The modules contained in the single cell analysis pipeline are the following:

  1. Images: Simply drag and drop the image files of a time series in the Images module. Make sure that the name of all the files in the dataset do include the name of the channel where the fluorescence was measured (e.g., GFP) and the increasing consecutive numbers for time-lapse microscopy images (e.g., GFP_72h).

  2. NamesAndTypes: Select the rule criteria with an expression contained in the name of all the files in the dataset, in this case, indicating the channel of the fluorescence (e.g., GFP). For each file of the dataset it creates an image called “Sensor”.



    Figure 4. CellProfiler image analysis pipeline for single cell tracking of flavivirus infected BHK-21/FlaviA-GFP reporter cells


  3. CorrectIlluminiationCalculate (Illumination function calculation: Regular): From the “Sensor” image, this module calculates a regular illumination correction function and creates an image with enhanced contrast, named “IllumSensor”.

  4. CorrectIlluminiationCalculate (Illumination function calculation: Background): From the “Sensor” image, this module calculates a background-based illumination function called “IllumSensorbackground”.

  5. RescaleIntensity: Converts the output image of the regular illumination function (“IllumSensor”) to an image with rescaled intensity (“RescaleSensor”).

  6. CorrectIlluminationApply: This module applies the “IllumSensorbackground” correction function to the “RescaleSensor” image and generates an enhanced image named “CorrSensor”.

  7. CorrectIlluminationApply: This second module of correction works on the original “Sensor” image, applies the “IllumSensorbackground” correction function but keeping the original intensity values and generates an output image called “mSensor”.

  8. IdentifyPrimaryObjects: This module works with the enhanced image “CorrSensor” to identify the objects Cells. You may modify the Typical diameter to obtain the correct cell segmentation with other cell lines or parameters.

  9. MeasureObjectIntensity: Measures the intensities of the identified objects Cells but on the image with the original intensity values only corrected by illumination (“mSensor”).

  10. TrackObjects: This module tracks cells over the time-lapse microscopy and generates an output image called “TrackedCells”.

  11. GraytoColor, OverlayOutlines, SaveImages: These modules create and save color images with the outlines of the primary objects identification for documentation and validation of the cell segmentation by the researcher’s eye and criteria.

  12. ExportToSpreadsheet: Saves the selected measurements to an excel spreadsheet (e.g., Integrated intensity).

    Note: Together with this protocol we supply the Zip file “SingleCellsTracking pipeline and dataset” which contains the applied pipeline (“SingleCellsTracking.cpproj”) and an example dataset composed of a single input image to run the pipeline (“Input_image_GFP”) and two output files to corroborate the expected results (“Output_image_GFP” and “Infected_Cells”).


  1. Kinetic plaque assay in reporter cells

    The identification and tracking of viral plaques were performed using two different pipelines programmed in CellProfiler. For single-plaque recognition use our “PlaqueIdentification.cpproj” pipeline (Figure 5) to generate images such as those depicted in the upper panel of Figure 3B. To track the plaques over a time series and determine the cell counts for every recognized plaque, first apply the “PlaqueIdentification.cpproj” pipeline to generate an image with the identified plaques and then use that image and the “PlaqueTracking.cpproj” pipeline (Figure 6) to obtain tracking images such as those showed in the lower panel of Figure 3B. Depending on the cell type and/or density, some modifications to several parameters may be done for an optimal plaque identification using the option Start Test Mode and running each module step by step. The modules of the above mentioned pipelines are the following:



    Figure 5. CellProfiler image analysis pipeline for flaviviral plaques identification in BHK-21/mNeptune reporter cells


    Figure 6. CellProfiler image analysis pipeline for flaviviral plaques tracking in BHK-21/mNeptune reporter cells


    Plaque identification pipeline

    1. Images: Simply drag and drop the image files of a time series in the Images module. Make sure that the name of all the files in the dataset do include the name of the channel where the fluorescence was measured (e.g., mNeptune) and the increasing consecutive numbers for time-lapse microscopy images (e.g., mNeptune_72h).

    2. NamesAndTypes: Select the rule criteria with an expression contained in the name of all the files in the dataset, in this case, indicating the channel of the fluorescence (e.g., mNeptune). For each file of the dataset it creates an image called “Sensor”.

    3. Crop: This is an optional module in case you need to remove part of the image (e.g., distance bars). It generates an output image named “CropSensor”.

    4. IdentifyPrimaryObjects: This module identifies the individual objects Nuclei from the “CropSensor” image. You may need to change the Typical diameter of objects or the Threshold strategy and its parameters to obtain an optimal identification of your cells.

    5. MeasureObjectNeighbors: Measures the number of neighbors for each Nuclei object within a specified Neighbor distance. You may need to modify this parameter to obtain a good optimal range of neighbor numbers to differentiate the plaque-belonging cells from the surrounding cells. This module creates an image called “ObjectNeighborCount”.

    6. FilterObjects: This module sets a threshold to identify plaque-belonging Nuclei objects using the range of number of neighbors calculated in the previous module. You may need to set this Minimum value according to the range of neighbors for an optimal identification of plaque belonging Nuclei objects. It generates the output objects FilteredNuclei.

    7. IdentifySecondaryObjects: Extends the above FilteredNuclei by a determined number of pixels to fill the gaps between the plaque-belonging cells in the “CropSensor” image. You may need to modify the Number of pixels by which to expand the primary objects in order to fill most of the gaps. This module identifies the output objects Cells.

    8. ConvertObjectstoImage: The expanded Cells objects constitute the basis to create a new image named “CellImage”.

    9. IdentifyPrimaryObjects: Uses the previous “CellImage” to identify the new Plaque objects. You may need to modify the range of Typical diameter of objects to obtain the correct plaque identification with images from different post-infection times, as shown in the colored frame Plaque, within the visual output generated by this module. In such an instance images must be analyzed one by one, like in the case of our example dataset.

    10. ConvertObjectstoImage: Converts the Plaque objects to an image called “PlaqueImage”.

    11. SaveImages: Uses the “PlaqueImage” to create and save an image called “Output_image_PreviousPlaques”, that constitutes one of the input images for the plaque tracking with the pipeline “PlaqueTracking.cpproj”.

    12. OverlayObjects: This module fuses the “CropSensor” image with the Plaque objects to make a composite image named “OverlayImage”.

    13. SaveImages: Creates an image called “Output_image_OverlayPlaques” based on the “OverlayImage”.

      Note: Together with this protocol we supply the Zip file “PlaqueIdentification tracking pipeline and dataset” which contains the applied pipeline (“PlaqueIdentification.cpproj”) and an example dataset composed of two input images to run the pipeline and practice the required adjustments (“Input_image_mNeptune_72h” and “Input_image_mNeptune_96h”) and four output files to corroborate the expected results (“Output_image_PreviousPlaques_72h”, “Output_image_PreviousPlaques_96h”, “Output_image_OverlayPlaques_72h”, and “Output_image_OverlayPlaques_96h”).


    Plaque tracking pipeline

    1. Images: Simply drag and drop the files of the plaque assay fluorescence images (e.g., “Input_image_mNeptune_72h”) and the output images created with the previous Plaque identification pipeline (e.g., “Output_image_PreviousPlaques_72h”). Make sure that the name of the plaque assay fluorescence images do include the name of the channel where the fluorescence was measured (e.g., mNeptune) and the increasing consecutive numbers for time-lapse microscopy images (e.g., mNeptune_72h).

    2. NamesAndTypes: Select the rule criteria with an expression contained in the name of all the plaque assay fluorescence images, in this case, indicating the channel of the fluorescence (e.g., mNeptune). For each fluorescence image it creates an image called “Sensor”.

    3. The modules 3-8 are the same as those described in the Plaque identification pipeline.

    4. IdentifySecondaryObjects: This module identifies the new Plaque objects in the “CellImage” as secondary objects around the previously identified PreviousPlaques primary objects.

    5. TrackObjects: This module tracks the plaques over time using an overlap criteria across the time-resolved images and creates an output image named “TrackedPlaques”. You may adjust the Maximum distance to consider matches for an optimal plaque tracking.

    6. SaveImages: Saves the “Tracked_Plaques” image for your validation and final results.

    7. RelateObjects: This module correlates the new Plaque objects with the previously identified Nuclei objects.

    8. ExporttoSpreadSheet: This module exports to an excel spreadsheet the data of the number of cells (Nuclei objects) that compose every recognized plaque in all the analyzed images.

      Note: Together with this protocol we supply the Zip file “PlaqueIdentification tracking pipeline and dataset” which contains the applied pipeline (“PlaqueTracking.cpproj”) and an example dataset composed of four input images to run the pipeline (“Input_image_mNeptune_72h”, “Input_image_mNeptune_96h”, “Output_image_Previous Plaques_72h”, and “Output_image_PreviousPlaques_96h”) and three output files to corroborate the expected results (“Tracked_Plaques_72h”, “Tracked_Plaques_96h” and “Viral_Plaque”).

    Recipes

    1. Complete DMEM

      500 ml DMEM (Gibco)

      5 ml Antibiotic-antimycotic 100×

      5 ml (for 2%) or 50 ml (for 10%) of FBS

      Homogenize by hand rotation (20 times, gently to avoid formation of foam)

      Store at 4 °C. Stable for 4 months

    2. PEI solution (1 mg/ml)

      1 mg of PEI powder

      1 ml of HCl 0.2 M in distilled water

      Heat and shake at 60 °C, 600 rpm in a ThermoMixer® block

      Sterilize by filtration (0.2 µm pore size)

      Prepare 45 µl aliquots into 2 ml reaction tubes and store at -80° C. Stable for 4 months

    3. Polybrene solution (10 mg/ml)

      10.6 mg of hexadimethrine bromide powder

      Dissolve in 1 ml of destilled water

      Sterilize by filtration (0.2 µm pore size)

      Prepare 50 µl single use aliquots into 0.5 ml reaction tubes and store at -20 °C. Stable for 1 year

    4. Complete MEM

      500 ml MEM (Gibco)

      5 ml Sodium pyruvate 100 mM

      5 ml Antibiotic-antimycotic 100×

      5 ml (for 2%) or 50 ml (for 10%) of FBS

      Homogenize by hand rotation (20 times, gently to avoid formation of foam)

      Store at 4 °C. Stable for 4 months

    5. PBS 1% FBS

      45 ml PBS, pH 7.4

      5 ml complete MEM 10% FBS

      Store at 4 °C. Stable for 4 months

    6. Puromycin solution (10 mg/ml)

      10 mg of puromycin

      Dissolve in 1 ml of destilled water

      Sterilize by filtration (0.2 µm pore size)

      Prepare 20 µl single use aliquots into 1.5 ml light protection reaction tubes and store at -20 °C. Stable for 2 years

    7. FluoroBriteTM DMEM 2% FBS

      500 ml FluoroBriteTM DMEM (Gibco)

      5 ml GlutaMAXTM supplement

      5 ml Sodium pyruvate 100 mM

      5 ml Antibiotic-antimycotic 100×

      5 ml of FBS

      Homogenize by hand rotation (20 times, gently to avoid formation of foam)

      Store at 4 °C. Stable for 4 months

    8. Plaque media 2% FBS

      9.4 g MEM (Sigma)

      900 ml Milli-Q water

      Adjust pH to 4.0

      10 g Carboxymethylcellulose sodium salt

      Autoclave (121 °C at 100 kPa for 15 min)

      30 ml 7.5% sodium bicarbonate solution in Milli-Q water (sterilized by filtration – 0.2 µm pore size)

      10 ml GlutaMAXTM supplement

      10 ml Sodium pyruvate 100mM

      10 ml Antibiotic-antimycotic 100×

      20 ml of FBS

      Homogenize by hand rotation (20 times, gently to avoid formation of foam)

      Store at 4 °C. Stable for 4 months

    Acknowledgments

    We want to thank Dr. Jeanne A. Hardy from the Department of Chemistry, University of Massachusetts, for her kindness and scientific advice during our outstanding collaboration. We also want to acknowledge our funding sources at Universidad de Costa Rica (project VI-803-B9-505) and International Centre for Genetic Engineering and Biotechnology (Grant CRP/CRI18-02).

    Competing interests

    The authors declare that they do not have any conflicts of interests.

    References

    1. Arias-Arias, J. L., MacPherson, D. J., Hill, M. E., Hardy, J. A. and Mora-Rodriguez, R. (2020). A fluorescence-activatable reporter of flavivirus NS2B-NS3 protease activity enables live imaging of infection in single cells and viral plaques.J Biol Chem 295(8): 2212-2226.
    2. Balsitis, S. J., Coloma, J., Castro, G., Alava, A., Flores, D., McKerrow, J. H., Beatty, P. R. and Harris, E. (2009). Tropism of dengue virus in mice and humans defined by viral nonstructural protein 3-specific immunostaining. Am J Trop Med Hyg 80(3): 416-424.
    3. Freppel, W., Mazeaud, C. and Chatel-Chaix, L. (2018). Production, titration and imaging of Zika virus in mammalian cells. Bio-protocol 8(24): e3115.
    4. Gould, E. A. and Solomon, T. (2008). Pathogenic flaviviruses.Lancet 371(9611): 500-509.
    5. Hawley, T. S., Herbert, D. J., Eaker, S. S. and Hawley, R. G. (2004). Multiparameter flow cytometry of fluorescent protein reporters. Methods Mol Biol 263: 219-237.
    6. Hsieh, M. S., Chen, M. Y., Hsieh, C. H., Pan, C. H., Yu, G. Y. and Chen, H. W. (2017). Detection and quantification of dengue virus using a novel biosensor system based on dengue NS3 protease activity. PLoS One 12(11): e0188170.
    7. Kümmerer, B. M. (2018). Establishment and application of flavivirus replicons. Adv Exp Med Biol 1062: 165-173.
    8. Li, S. H., Li, X. F., Zhao, H., Deng, Y. Q., Yu, X. D., Zhu, S. Y., Jiang, T., Ye, Q., Qin, E. D. and Qin, C. F. (2013). Development and characterization of the replicon system of Japanese encephalitis live vaccine virus SA14-14-2. Virol J 10: 64.
    9. McFadden, M. J., Mitchell-Dick, A., Vazquez, C., Roder, A. E., Labagnara, K. F., McMahon, J. J., Silver, D. L. and Horner, S. M. (2018). A Fluorescent Cell-Based System for Imaging Zika Virus Infection in Real-Time.Viruses 10(2).
    10. Medin, C. L., Valois, S., Patkar, C. G. and Rothman, A. L. (2015). A plasmid-based reporter system for live cell imaging of dengue virus infected cells. J Virol Methods 211: 55-62.
    11. Medina, F., Medina, J. F., Colon, C., Vergne, E., Santiago, G. A. and Munoz-Jordan, J. L. (2012). Dengue virus: isolation, propagation, quantification, and storage. Curr Protoc Microbiol Chapter 15: Unit 15D 12.
    12. Schmid, B., Rinas, M., Ruggieri, A., Acosta, E. G., Bartenschlager, M., Reuter, A., Fischl, W., Harder, N., Bergeest, J. P., Flossdorf, M., Rohr, K., Höfer, T. and Bartenschlager, R. (2015). Live cell analysis and mathematical modeling identify determinants of attenuation of dengue virus 2'-o-methylation mutant. PLoS Pathog 11(12): e1005345.
    13. Soto-Garita, C., Somogyi, T., Vicente-Santos, A. and Corrales-Aguilar, E. (2016). Molecular Characterization of Two Major Dengue Outbreaks in Costa Rica. Am J Trop Med Hyg 95(1): 201-205.
    14. Tamura, T., Fukuhara, T., Uchida, T., Ono, C., Mori, H., Sato, A., Fauzyah, Y., Okamoto, T., Kurosu, T., Setoh, Y. X., Imamura, M., Tautz, N., Sakoda, Y., Khromykh, A. A., Chayama, K. and Matsuura, Y. (2018). Characterization of recombinant flaviviridae viruses possessing a small reporter tag. J Virol 92(2).
    15. Tiscornia, G., Singer, O. and Verma, I. M. (2006). Production and purification of lentiviral vectors. Nat Protoc 1(1): 241-245.
    16. Xie, X., Zou, J., Shan, C., Yang, Y., Kum, D. B., Dallmeier, K., Neyts, J. and Shi, P. Y. (2016). Zika virus replicons for drug discovery. EBioMedicine 12: 156-160.

简介

[摘要]本属黄病毒家族中的黄病毒包括医学重要性许多病毒种类,如黄热病病毒(YFV),寨卡病毒(ZIKV)和登革热病毒(DENV),等等。目前,黄病毒感染细胞的鉴定是基于病毒蛋白的免疫标记,重组报告子复制子和病毒基因组的应用,或黄病毒蛋白酶NS2B-NS3活性的基于细胞的分子报告子的使用。在后者中,我们的黄病毒可激活的GFP和mNeptune报道分子含有通过接头连接到荧光蛋白的淬灭肽(QP),该接头由黄病毒NS2B - NS3蛋白酶(AAQRRGRIG)的切割位点组成。当病毒蛋白酶切割接头时,淬灭肽被去除,并且荧光蛋白采用促进荧光的构象。在这里,我们提供了用于表达,选择和实施表达黄病毒基因编码分子报告子的稳定BHK-21细胞的详细协议,适用于通过活细胞成像监测病毒感染。我们还将描述图像分析过程并提供所需的软件管道。我们的报告细胞允许通过活细胞成像对黄病毒的参考菌株和天然菌株实施单细胞感染动力学以及噬菌斑测定。

图形摘要:

黄病毒感染实时成像的报告基因BHK-21细胞的产生与实施工作流。


[背景]黄病毒代表了正在引起并正在重新出现的全球性威胁,可能引起动物和人类疾病,包括许多与医学有关的病毒,例如黄热病病毒(YFV),西尼罗河病毒(WNV),日本脑炎病毒(JEV),登革热病毒(DENV),并兹卡六RUS(ZIKV),等等(摹·乌尔德·所罗门,2008) 。目前,黄病毒感染细胞的检测基于病毒蛋白的抗体标记(B alsitis等,2008),重组报告子复制子和病毒基因组的使用(B Li等。,2013;Schmid等。,2015;谢等。,2016年; 田村等。,2017; Kümmerer,2018 ) 或黄病毒NS2B- NS3蛋白水解活性的基因编码分子报道分子的应用(Medin ,CL等人,2015; H sieh等人,2017; McFadden等人,2018)。免疫标记意味着固定和通透化,这阻碍了它们在活细胞中的研究实施。报告基因复制子和病毒基因组适用于活细胞成像分析,但它们仅限于主要来源于参考菌株的特定分子克隆,因此不适用于临床分离株或天然病毒株。在这种情况下,黄病毒蛋白酶的基于细胞的分子报告分子构成了通过活细胞成像研究天然黄病毒株的理想替代方法。根据我们最近发布的黄病毒可激活的GFP(FlaviA -GFP)和黄病毒可激活的mNeptune (FlaviA-mNeptune )报道分子(A rias-Arias等,2020),在此我们详细描述了生成,选择,稳定转导的报告基因BHK-21细胞用于单细胞和病毒斑块中黄病毒感染的实时成像。此外,我们为基于软件的图像分析方法提供了理论基础,以证明该报告细胞系对单细胞和病毒斑块跟踪的功能。此外,我们还包括优化的CellProfiler分析管道,用于使用此或其他基于单元格的报告子的研究。我们的方法代表了第一个基于荧光可激活细胞的报告系统,用于通过活细胞成像监测黄病毒的参考株和天然株(如DENV,ZIKV和YFV)的感染动力学。

关键字:黄病毒属, 荧光, NS2B-NS3, 蛋白酶, 活细胞成像, 报告细胞, 噬斑试验, 图象分析

材料和试剂

1.细胞培养烧瓶,75厘米2 (格雷纳生物一,蜂星® ,目录号:658175)     

2.细胞培养皿,100/20毫米(格雷纳生物一,蜂星® ,目录号:664160)     

3. 2 ml反应管(Greiner Bio-One,目录号:623201)     

4. 3毫升无菌注射器(Ultident Scientific,目录号:BD-309657)     

5.针头式过滤器0.2微米的亲水性聚醚砜,32mm直径(帕尔,的Acrodisc ® ,目录号:4652)     

6. 10毫升无菌注射器(Ultident Scientific,目录号:BD-302995)     

7.注射器过滤0.45微米醋酸纤维素亲水性,有28毫米直径的(赛多利斯,Minisart ® ,目录号:16555)     

8. 15ml锥形管(格雷纳生物一,蜂星® ,目录号:188271)     

9. 0.5 ml反应管(Greiner Bio-One,目录号:667201)     

10.溴己二甲胺(Merck,Sigma-Aldrich,目录号:H9268) 

11. 48孔细胞培养板(Greiner生物一,蜂星® ,目录号:677180) 

12. 1.5 ml反应管(Greiner Bio-One,目录号:616201) 

13. 12孔细胞培养板(Greiner生物一,蜂星® ,目录号:665180) 

14. 1.5 ml光保护反应管(Greiner Bio-One,目录号:616283) 

15.细胞培养烧瓶,25厘米2 (格雷纳生物一,蜂星® ,目录号:690175) 

16. 6孔细胞培养板(Greiner生物一,蜂星® ,目录号:657160) 

17. 96-孔黑色细胞培养板(Greiner生物一,μCLEAR ® ,目录号:655096) 

18. 24孔细胞培养板(Greiner生物一,蜂星® ,目录号:662160 ) 

19. HEK 293T细胞(ATCC,目录号:CRL-3216) 

20.质粒: 

pLenti - FlaviA -GFP-puro (来自Jorge L. Arias-Arias的礼物,Addgene质粒#140088)


pLenti -CMV- FlaviA - mNeptune -puro (Jorge L. Arias-Arias的礼物,Addgene质粒#140091)


pMD2.G(Didier Trono的礼物,Addgene质粒#12259)


psPAX2(Didier Trono的礼物,Addgene质粒#12260)


21.含100 µg / ml氨苄青霉素的LB琼脂平板(Merck,Sigma-Aldrich,目录号:L5667) 

22. LB汤(米勒)(默克,西格玛奥德里奇,目录号:L2542) 

23. 100 mg / ml氨苄青霉素溶液(Merck,Sigma-Aldrich,目录号:A5354) 

24. NucleoSpin质粒微型试剂盒(Macherey -Nagel,目录号:740588.50) 

25. DMEM,高葡萄糖,GlutaMAX TM ,丙酮酸(Thermo Fisher Scientific,Gibco ,目录号:10569044) 

26.抗生素-抗真菌药100 × (Thermo Fisher Scientific,Gibco ,目录号:15240062) 

27.合格的胎牛血清(FBS),经热灭活(Thermo Fisher Scientific,Gibco ,目录号:10438-026) 

28.线性亚乙基聚乙烯亚胺(PEI),MW 25000,转染等级(Polysciences ,PEI 25K TM ,目录号:23966-1) 

29. UltraPure TM不含DNase / RNase的蒸馏水(Thermo Fisher Scientific,Invitrogen,目录号:10977015) 

30.盐酸36.5-38.0%,生物试剂(Merck,Sigma-Aldrich,目录号:H1758) 

31. BHK-21 [C-13](ATCC,目录号:CCL-10) 

32. MEM,GlutaMAX TM补充品(Thermo Fisher Scientific,Gibco ,目录号:41090101)。 

33.丙酮酸钠,100 mM的(热Fisher Scientific公司,Gibco公司,目录号:11360070)。 

34. PBS,pH 7.4中(热Fisher Scientific公司,Gibco公司,目录号:10010023)。 

35. TrypLE TM表达酶(1 × ),无酚红(Thermo Fisher Scientific,Gibco ,目录号:12604013) 

36.嘌呤霉素二盐酸盐从链霉菌alboniger (默克,Sigma-Aldrich公司,目录号:P8833) 

37.临床分离株DENV-2 / CR / 13538/2007 (哥斯达黎加,卡塔哥,Costarricense deInvestigaciónyEnseñanzaenNutricióny Salud研究所)(S oto-Garita等人,2016年) 

38.疫苗株YFV / US / 17D / 1937(赛诺菲巴斯德,YF-VAX ® )   

39. FluoroBrite TM DMEM(Thermo Fisher Scientific,Gibco ,目录号:A1896701) 

40. GlutaMAX TM补充剂(Thermo Fisher Scientific,Gibco ,目录号:35050061) 

41.最低必需中型鹰牌AutoMod TM (默克,西格玛奥德里奇,目录号:M0769) 

42.羧甲基纤维素钠盐(Merck,Sigma-Aldrich,目录号:C4888) 

43.碳酸氢钠(Merck,西格玛奥德里奇,目录号:S5761) 

44.完整的DMEM (请参阅食谱) 

45. PEI溶液(1毫克/毫升)(请参阅食谱) 

46.聚乙烯溶液(10毫克/毫升)(请参阅食谱) 

47.完整的MEM (请参阅食谱) 

48. PBS 1%FBS (请参阅食谱) 

49.嘌呤霉素溶液(10毫克/毫升)(请参阅食谱) 

50. FluoroBrite TM DMEM 2%FBS (请参阅食谱) 

51. 2%FBS斑块培养基(请参见食谱) 


设备

生物安全柜(ESCO,Labculture ® II级,A2型,目录号:LA2-3A2-E)
CO 2培养箱(Thermo Scientific,型号:Forma Series II,目录号:3110)
4°C冰箱(Thermo Scientific,价值实验室,目录号:20LREETSA)
-20°C冰箱(Thermo Scientific,Value Lab,目录号:20LFEETSA)
-80°C冰柜(三洋,VIP系列,目录号:MDF-U32V)
水浴(PolyScience ,目录号:WBE20A12E)
离心机(Eppendorf,型号:5810)
微量离心机(Eppendorf,型号:5418)
移液器(T hermo Scientific,Finnpipette TM F2 GLP试剂盒,目录号:4700880)
移液器(T hermo Scientific,S1,目录号:9511)
血细胞计数器(Boeco ,Neubauer改进,目录号:BOE 13) 
摇动培养箱(Shel Lab,目录号:SSI3)
恒温® C座仪(Eppendorf,目录号:5382000015 )
涡旋混合器(T hermo Scientific,MaxiMix TM ,目录号:M16715Q)
紫外线交联剂(UVP,CL-100,目录号:UVP95017401)
NanoDrop TM 2000分光光度计(Thermo Scientific,目录号:ND-2000)
蒸汽灭菌器(大和号,目录号:SQ510)
净水系统(Merck,Milli -Q优势A10)
倒置显微镜(尼康,日食,货号:TS100)
流式细胞仪(BD Biosciences,BD Accuri TM C6)
细胞分选仪(BD Biosciences,BD FACSJazz TM )
自动荧光显微镜(Biotek ,Lionheart FX)具有:
CO 2气体控制器(Biotek ,目录号:1210012)


ħ umidity室(Biotek的,目录号:1450006)


4 ×物镜(Biotek ,目录号:1220519)


20 ×物镜(Biotek ,目录号:1220517)


GFP过滤器立方体(Biotek ,目录号:1225101)


465 nm LED立方体(Biotek ,目录号:1225001)


Cy5过滤器立方体(Biotek ,目录号:1225105)


623 nm LED立方体(Biotek ,目录号:1225005)

软件

1. Gen5 Image +(Biotek ,https : //www.biotek.com )      


2. CellProfiler 4.0(Broad Institute,https: //cellprofiler.org/releases )      

程序

慢病毒载体的组装和滴定
部件


1.准备下列标准分子生物学程序质粒股票(https://www.jove.com/v/5062/plasmid-purification)和协议由制造商提供的的NucleoSpin质粒微型试剂盒(马歇雷-Nagel)。     

2.用8 ml DMEM 10%FBS(配方1)在100/20 mm细胞培养皿中手动接种6,000,000个HEK 293T细胞,并在37°C和5%CO 2下孵育过夜,以达到70-80%融合度。有关播种细胞的详细步骤,请参阅Freppel等人的生物协议论文,2018年(参考文献3)。     

3.使用移液器,除去并丢弃培养皿中的所有DMEM 10%FBS(〜8 ml),然后加入4 ml新鲜的DMEM 2%FBS(配方1),以更换培养基。     

4.如下准备转染混合物:      


悬浮液A:45 µl PEI 1 mg / ml(配方2 )+ 955 µl未补充的DMEM(不含FBS和抗真菌药)。 


悬浮液B:6微克pLenti -弗拉维亚-GFP-PURO或pLenti -CMV-弗拉维亚- mNeptune -puro + 6微克pMD2.G + 6微克psPAX2和带来至1,000微升与未补充的DMEM。


将悬浮液A与B混合,并在室温下孵育15分钟。


5.将2 ml转染混合物添加到细胞中(逐滴),并在37°C和5%CO 2下孵育过夜。     

6.用6 ml新鲜的DMEM 2%FBS代替培养基,并在37°C和5%CO 2下孵育48小时。     

7.收集培养上清液并过滤(孔径为0.45-μm)以清除细胞和碎片。使用蛋白质粘附力较低的过滤器,例如醋酸纤维素或聚醚砜(PES)。不要使用硝酸纤维素滤膜,因为它们可能会结合慢病毒颗粒。       

8.准备500 µl等分试样,并在-80°C下储存。     


滴定法


1.如上所述,在具有500μl/孔MEM 2%FBS(配方4)的48孔细胞培养板上,每孔接种50,000 BHK-21细胞,并在37°C和5%CO 2下孵育过夜。准备额外的孔以用作对照细胞。     

2.在水浴中(37°C)解冻500 µl慢病毒等分试样之一,并以5 µg / ml的终浓度添加聚乙烯(配方3)。     

3.准备10倍系列稀释液(10 -1 -10 -6慢病毒库存的)在1.5ml反应管中。将50 µl慢病毒颗粒开始在450 µl 2%FBS + 5 µg / ml聚乙烯(10 -1 )的DMEM中混合。将试管涡旋5 s,然后将50 µl 10 -1慢病毒稀释液与450 µl DMEM 2%FBS + 5 µg / ml聚乙烯(10 -2 )混合。对其他稀释液(10 -3 -10 -6 )重复此步骤,并在室温下孵育15分钟。在稀释液之间更换吸头,以避免交叉污染。有关10倍系列稀释液制备的详细图形说明,请参阅Freppel等人于2018年发表的生物协议论文(参考文献3)。     

4.用150 µl /孔的未稀释慢病毒原液及其各系列稀释液替换细胞培养基。向对照细胞中加入150 µl /孔的DMEM 2%FBS + 5 µg / ml聚乙烯。为避免交叉污染,从对照开始,依次稀释(从高到低稀释),再从未稀释的慢病毒原种开始,依次接种样品。     

5.在25°C下以300 × g离心2 h以进行病毒吸附。     

6. ř E放置用500μl/孔的DMEM 2%FBS并孵育48小时接种物在37℃和5%CO 2 。     

7.弃去培养基,用100 µl /孔的PBS洗涤细胞一次,添加100 µl /孔的TrypLE TM表达(无酚红),在37°C下孵育5分钟,然后用400 µl /孔重悬细胞。 PBS 1%FBS的孔(配方5 )。     

8.根据报告蛋白的基础背景,并与未转导的对照细胞进行比较(图1),通过流式细胞仪确定GFP + (488 nm激光-530/30 nm滤光片)或mNeptune +(640慢病毒种子的不同稀释度感染的样品中存在1 nm激光-675/25 nm滤光片)细胞。有关荧光蛋白流式细胞术的详细步骤,请参阅Hawley等人(2004年)的协议(参考文献5)。       

9.使用可检测的转导细胞的较高稀释度数据,使用下式计算以转导单位每毫升(TU / ml)为单位的生物效价:     

TU / ml =(P×N / 100×V)×1 / DF

其中P = GFP +或mNeptune +细胞的百分比,N =转导时的细胞数量= 50,000,V =每孔的接种量= 0.15 ml,DF =稀释因子= 1(未稀释),10 –1 (稀释1/10) ),10 –2 (稀释1/100)等(T iscornia等,2006)。

记者细胞系的生产和选择
生产


1.如上所述,在具有1 ml /孔的MEM 2%FBS的12孔细胞培养板上,每孔接种100,000 BHK-21细胞,并在37°C和5%CO 2下孵育过夜。为对照细胞准备一个额外的孔。      


2.解冻在水浴(37℃)携带遗传构建编纂为任一慢病毒颗粒的500个微升等分试样的库存中的一个弗拉维亚-GFP或Flavia的-mNeptune记者和添加聚brene以5μg最终浓度/毫升      


3.根据先前计算的以TU / ml为单位的生物学滴度,以500的感染率(MOI)为1 (每细胞1 TU )制备500微升/孔的慢病毒接种物。如100,000个细胞每孔铺板在manteinance培养基(MEM 2%FBS),100000 TU必须在MEM 2%FBS + 5微克/毫升稀释聚凝胺至500的最终体积微升。在室温下孵育15分钟。      


4.用500 µl /孔的len tiviral接种物替换细胞培养基。向对照细胞中加入500 µl /孔的MEM 2%FBS + 5 µg / ml聚乙烯。      


5.在25°C下以300 × g离心2 h以进行病毒吸附。      


6.用1 ml /孔的MEM 2%FBS代替接种物,并在37°C和5%CO 2下孵育48小时。      


7.基于报告蛋白的基础背景,通过荧光显微镜在绿色/ GFP(FlaviA -GFP)和远红/ Cy5(FlaviA-mNeptune )通道中监测转导的有效性。      

选拔


1.为选择转导细胞的抗生素,用每孔750 µl含8 µg / ml嘌呤霉素的MEM 10%FBS代替培养基(R ecipe 6),并在37°C和5%CO 2下孵育过夜。对对照细胞进行相同的处理。     

2.用1 ml /孔的MEM 10%FBS含4 µg / ml的嘌呤霉素替换转导和对照细胞的培养基。在光学显微镜下(通常24-48小时)证明在37°C和5%CO 2下孵育直至对照细胞的100%死亡率。       

3.用1 ml /孔的MEM 10%FBS + 0.5 µg / ml嘌呤霉素替换所选细胞的培养基,并在37°C和5%CO 2下孵育,直至达到80-90%的融合度。     

4.将选定的细胞加入5 ml MEM 10%FBS + 0.5 µg / ml嘌呤霉素的25cm²培养瓶中,并在37°C和5%CO 2下孵育,直至达到80-90%的融合度(https:// www.jove.com/v/5052/passaging-cells)。     

5.对于FACS选择,丢弃培养基,用1 ml PBS洗涤细胞一次,加入500 µl TrypLE TM表达(无酚红),在37°C下孵育5分钟,然后用1 ml重悬细胞。每毫升PBS 1%FBS。     

6.用血细胞计数器(https://www.jove.com/v/5048/using-a-hemacytometer-to-count-cells)计数细胞,并以1,000,000个细胞/ ml在PBS 1中制备2 ml细胞悬液。 %FBS 。     

7.将细胞悬液吸入细胞分选仪。对于FlaviA -GFP报道分子,使用FL1检测器(488 nm激光-530/30 nm滤光片)分离出至少两个具有不同但均一水平的报道者基础背景的细胞亚群(图1)。将相同的步骤应用于FlaviA-mNeptune报告基因,但使用FL4检测器(640 nm激光-675/25 nm滤光片)。     

注意:FlaviA -GFP和FlaviA-mNeptune报告基因的基础背景使其对细胞表达水平敏感:如果表达过高,背景将掩盖报告基因激活后产生的信号。如果表达太低,则背景中将没有足够的报告信号。



图1. BHK-21 / FlaviA -GFP稳定细胞的FACS分析。A.直方图显示由于FlaviA -GFP报告蛋白的基础背景,野生型和报告BHK-21细胞之间绿色荧光(FL1检测器530/30 nm滤光片)之间的差异。B.散点图显示了具有不同水平的FlaviA- GFP报告基因的稳定BHK-21细胞群体的异质性。为了选择最佳的报告细胞(最高信噪比),必须分离至少两个具有不同(低,中或高)但均一水平的报告者基础背景的细胞亚群,并通过活细胞成像进行测试在黄病毒感染后。

8.小号EED孤立小区suppopulations在48孔细胞培养板的不同孔用1ml /孔的MEM 10个%FBS + 0.5微克/毫升PU霉素孵育在37℃和5%CO 2 ,直到达到汇合度为80-90%。     

9.通道所选择的细胞的6 -孔细胞培养板用3ml MEM 10%FBS的+ 0.5微克/毫升嘌呤霉素孵育在37℃和5%CO 2 ,直到达到80-90%汇合。     

10.根据步骤C中所述的方案,通过活细胞成像黄病毒感染动力学测试不同的分离细胞亚群。 

注意:最好的报告细胞是黄病毒感染后具有最高信噪比的细胞。荧光信噪比的计算方法是,在相同的接种后时间,将用感染性病毒处理过的报告细胞的信号除以用紫外线灭活的病毒处理过的报告细胞所产生的噪声。

通过活细胞成像在报告细胞中的感染动力学
1.准备和滴度的黄病毒你选择的种子(例如,DENV,ZIKV,或YFV)根据tostandard病毒学方法(麦地那,F.等人,2012; ˚F reppel 。等人,2018) 。为了使病毒灭活,将200 µl /孔的黄病毒种子放入24孔细胞培养板中,移开盖子,并以400,000 µJ / cm 2的能量将5个周期的紫外线(254 nm)暴露于紫外线交联剂中。在两次循环之间,用手摇动平板5秒钟。      


2.如上所述,在含100 µl /孔MEM 2%FBS的µClear黑色96孔板中每孔接种15,000 BHK-21 / FlaviA -GFP稳定细胞,并在37°C和5%CO 2下孵育过夜。      


3.根据以斑块形成单位(PFU)/ ml计算的黄病毒种子滴度,以低MOI(0.1至0.25)制备50 µl /孔的接种物。如15,000个细胞每孔在维持培养基铺板,1之间,500和3 ,750个PFUS必须在MEM 2%FBS稀释至50的最终体积微升/孔。乘以要接种的孔总数,并准备一个接种悬液。同样,准备紫外线灭活接种悬浮液。      


4.用50 µl /孔的感染性或紫外线灭活的黄病毒接种物替换细胞培养基,并在37°C和5 %CO 2中孵育2 h以进行病毒吸附。每15分钟用双手摇动平板5秒钟。      


5.用150 µl /孔的FluoroBrite TM DMEM 2%FBS(配方7)替换接种物,并在37°C和5%CO 2下孵育动力学所需的时间(例如120 h)到自动荧光显微镜中。向周围的孔中加入200 µl /孔的PBS,以避免干燥。      


6.使用您的显微镜的软件(例如,的Gen5图片+),节目中的图像采集与4 ×或20 ×物镜在绿色/ GFP通道在去播下感染后时间(图2A)。      


注意:图像的采集参数(激发强度,曝光时间和相机增益)根据通过FACS分离的特定报告细胞亚群而变化。务必包括额外的控制室孔,以调整采集参数,因为过度暴露于激发光可能会损害实验条件的细胞。我们的建议是将这些参数设置为在动力学的第一张图像中仅能感知到报告蛋白背景信号的水平,以增加在被黄病毒蛋白酶激活后报告基因荧光的动态范围。



图2.通过活细胞成像在报告BHK-21细胞中的DENV-2感染动力学。A.将表达FlaviA -GFP报告基因的稳定BHK-21细胞感染性或UV灭活的DENV-2 13538以低MOI接种0.1,并在指定时间段通过活细胞成像捕获。放大倍数为40 × ,比例尺= 100 µm。B.使用CellProfiler 4.0软件对感染动力学进行图像分析,可以根据报告的荧光随时间推移显示单个细胞(图中的每个彩色线对应于单个细胞)。黑色虚线表示细胞荧光的平均值。 


活细胞成像在报告细胞中进行动力学斑测定
1.如上所述,在含100 µl /孔MEM 10%FBS的µClear黑色96孔板中,每孔接种25,000 BHK-21 / FlaviA-mNeptune稳定细胞,并在37°C和5%CO 2下温育。     

2.在1.5 ml反应管中准备黄病毒种子的10倍系列稀释液(10 -1 -10 -6 )。将50 µl黄病毒种子开始混合在450 µl MEM 2%FBS(10 -1 )中。将试管涡旋5 s,然后将50 µl 10 -1黄病毒稀释液与450 µl MEM 2%FBS(10 -2 )混合。对其他稀释液(10 -3 -10 -6 )重复此步骤。在稀释液之间更换吸头,以避免交叉污染。有关10倍系列稀释液制备的详细图形说明,请参阅Freppel等人于2018年发表的生物协议论文(参考文献3)。     

3.用黄病毒种子每次系列稀释的50 µl /孔替换细胞培养基。用50 µl紫外线灭活的黄病毒种子接种对照孔。在37°C和5%CO 2下孵育2小时,以进行病毒吸附。每15分钟用双手摇动平板5秒钟。      


4.用150 µl /孔的噬菌斑培养基2%FBS(配方8)替换接种物,并在37°C和5%CO 2下孵育120 h到自动荧光显微镜中。向周围的孔中加入200 µl /孔的PBS,以避免干燥。      


5.使用您的显微镜的软件(例如,的Gen5图片+),节目中的图像采集(整个阱的蒙太奇)与4 ×在期望的感染后时间远红/ Cy5通道目标(图3A) 。      


注意:图像的采集参数(激发强度,曝光时间和相机增益)根据通过FACS分离的特定报告基因细胞的变化而变化。务必包括额外的控制室孔以调整采集参数,因为过度暴露于激发光可能会损害实验条件下的细胞。我们的建议是将这些参数设置为在动力学的第一张图像中仅能感知到报告蛋白背景信号的水平,以增加在被黄病毒蛋白酶激活后报告基因荧光的动态范围。



图3.通过活细胞成像在报告者BHK-21细胞中进行YFV动力学噬菌斑测定。A.用FlaviA-mNeptune报告基因的稳定BHK-21细胞接种感染性或紫外线灭活的YFV 17D的十倍稀释液(10 -1 -10 -6 )。加入2%FBS噬菌斑培养基后,在指定的时间通过活细胞成像捕获平板的整个孔。放大倍数为40 × ,比例尺= 1,000 µm。B.用软件CellProfiler 4.0对动力学噬菌斑测定进行图像分析,可以鉴定单个病毒噬菌斑(上图)并随时间追踪这些噬菌斑(下图)。对于得到的结果有更深的例证我们的动能斑块的检测,请观看活细胞成像v IDEO (视频1 )。



视频1.报告者BHK-21细胞的ZIKV噬菌斑测定动力学






数据分析

A.报告细胞中的感染动力学      


使用我们的CellProfiler管道进行单细胞分析(“ SingleCellsTracking.cpproj ” ,图4 ),根据感染细胞的荧光进行单细胞跟踪(图2B )。要执行此分析,只需将图像拖放到“图像”模块中,然后按“分析图像” 。为了修改适用于您的单元格和条件的管道,请转到“开始测试模式” ,调整模块中描述的参数(一个接一个),然后按“步骤”以查看应用于特定模块的修改的输出。准备好所有修改后,按Exit Test Mode并进入Analyze Images 。单细胞分析管道中包含的模块如下:


1.图像:只需将一个时间序列的图像文件拖放到“图像”模块中。确保数据集中所有文件的名称均包含测量荧光的通道的名称(例如GFP)和延时显微镜图像的连续编号(例如GFP_72h)。      


2. NamesAndTypes :在数据集中所有文件的名称中包含一个表达式,选择规则标准,在这种情况下,指示荧光的通道(例如GFP)。对于数据集的每个文件,它都会创建一个名为“ Sensor”的图像。      



图4 。CellProfiler图像分析管道可用于单细胞追踪黄病毒感染的BHK-21 / FlaviA -GFP报告基因细胞

3. CorrectIlluminiationCalculate (照明功能计算:常规):此模块从“传感器”图像中计算常规照明校正函数并创建具有增强对比度的图像,称为“ IllumSensor ”。      


4. CorrectIlluminiationCalculate (照明功能计算:背景):该模块从“传感器”图像中计算基于背景的照明功能,称为“ IllumSensorbackground ”。      


5. RescaleIntensity :将常规照明功能的输出图像(“ IllumSensor ”)转换为具有重新缩放强度的图像(“ RescaleSensor ”)。      


6. CorrectIlluminationApply :此模块将“ IllumSensorbackground ”校正功能应用于“ RescaleSensor ”图像,并生成名为“ CorrSensor ”的增强图像。      


7. CorrectIlluminationApply :第二个校正模块适用于原始“传感器”图像,应用“ IllumSensorbackground ”校正功能,但保留原始强度值,并生成称为“ mSensor ”的输出图像。      


8. IdentifyPrimaryObjects :此模块与增强型图像“ CorrSensor ”一起使用,以标识对象Cells 。您可以修改“典型”直径,以使用其他细胞系或参数获得正确的细胞分割。      


9. MeasureObjectIntensity :测量已识别对象单元格的强度,但在图像上具有仅通过照明(“ mSensor ”)校正的原始强度值。      


10. TrackObjects :此模块在延时显微镜下跟踪细胞,并生成称为“ TrackedCells ”的输出图像。 

11. GraytoColor ,OverlayOutlines ,SaveImages :这些模块创建并保存带有主要对象识别轮廓的彩色图像,以供研究人员的眼睛和标准进行文档记录和细胞分裂验证。 

12. ExportToSpreadsheet :将选定的度量保存到excel电子表格(例如,Integrated强度)。 

注意:与该协议一起,我们提供了一个Zip文件“ SingleCellsTracking管道和数据集” ,其中包含应用的管道(“ SingleCellsTracking.cpproj ”)以及一个由单个输入图像组成的示例数据集来运行管道(“ Input_image_GFP ”),两个输出文件以确认预期结果(“ Output_image_GFP ”和“ Infected_Cells ”)。

B.报告细胞中的动力学噬斑测定      


病毒斑块的鉴定和追踪是通过在CellProfiler中编程的两个不同管道进行的。对于单斑块识别,请使用我们的“ PlaqueIdentification.cpproj ”管道(图5)来生成图像,如图3B上面板所示。要在一个时间序列上跟踪斑块并确定每个识别出的斑块的细胞计数,请首先应用“ PlaqueIdentification.cpproj ”管道生成带有已识别斑块的图像,然后使用该图像和“ PlaqueTracking.cpproj ”管道(图6)获得跟踪图像,例如图3B下部面板中所示的那些图像。根据细胞类型和/或密度,可以使用“开始测试模式”选项并逐步运行每个模块来对几个参数进行一些修改,以实现最佳的噬菌斑识别。上述管道的模块如下:



图5.用于在BHK-21 / mNeptune报告细胞中鉴定黄病毒斑块的CellProfiler图像分析管线



图6.用于BHK-21 / mNeptune报告细胞中黄病毒斑块追踪的CellProfiler图像分析管线

斑块识别管线


1.图像:只需将一个时间序列的图像文件拖放到“图像”模块中。确保所有数据集中的文件名确实包括在该通道的名称测量荧光(例如,mNeptune )和时间推移显微镜图像增加连续编号(例如,mNeptune_72h)。     

2. NamesAndTypes :与包含在数据集中的所有文件,在这种情况下的名称的表达式选择规则标准,表明荧光(的信道例如,mNeptune )。对于数据集的每个文件,它都会创建一个称为“传感器”的图像。     

3.裁切:如果需要删除图像的一部分(例如,距离条),这是一个可选模块。它将生成名为“ CropSensor ”的输出图像。     

4. IdentifyPrimaryObjects :此模块从“ CropSensor ”图像中识别单个对象Nuclei 。您可能需要更改对象的“典型直径”或“阈值”策略及其参数,以获得对单元格的最佳标识。     

5. MeasureObjectNeighbors :测量在指定的Neighbor距离内每个Nuclei对象的邻居数。您可能需要修改此参数以获得邻居编号的最佳最佳范围,以区分属于噬菌斑的细胞与周围的细胞。该模块创建一个名为“ ObjectNeighborCount ”的图像。     

6. FilterObjects :此模块设置阈值,以使用上一个模块中计算的邻居数范围来识别斑块所属的Nuclei对象。您可能需要根据邻居的范围设置此最小值,以最佳识别斑块所属的Nuclei对象。它生成输出对象FilteredNuclei 。     

7. IdentifySecondaryObjects :将上述FilteredNuclei扩展确定的像素数,以填充“ CropSensor ”图像中属于斑块的单元格之间的间隙。您可能需要修改用于扩展主要对象的像素数,以填补大部分空白。此模块标识输出对象Cells 。     

8. ConvertObjectstoImage :展开的Cells对象构成了创建名为“ CellImage ”的新图像的基础。     

9. IdentifyPrimaryObjects :使用之前的“ CellImage ”来标识新的Plaque对象。您可能需要修改对象的“典型直径”范围,以使用此模块生成的可视输出中的不同感染后时间的图像来获得正确的斑块识别,如彩色帧“斑块”所示。在这种情况下,必须像我们的示例数据集一样,一张一张地分析图像。     

10. ConvertObjectstoImage :将Plaque对象转换为称为“ PlaqueImage ”的图像。 

11. SaveImages :使用“ PlaqueImage ”创建并保存一个名为“ Output_image_PreviousPlaques ”的图像,该图像构成了使用管道“ PlaqueTracking.cpproj ”进行斑块跟踪的输入图像之一。 

12. OverlayObjects :此模块将“ CropSensor ”图像与Plaque对象融合在一起,以生成名为“ OverlayImage ”的复合图像。 

13. SaveImages :创建名为“图像Output_image_OverlayPlaques ”的基础上“窗格在overlayImage ” 。 

注意:依此协议,我们提供了一个Zip文件“ PlaqueIdentification跟踪管道和数据集”,其中包含应用的管道(“ PlaqueIdentification.cpproj ”)以及一个示例数据集,该示例数据集由两个输入图像组成,以运行管道并进行必要的调整( “ Input_image_mNeptune_72h”和“ Input_image_mNeptune_96h”)以及四个输出文件以确认预期结果(“ Output_image_PreviousPlaques_72h”,“ Output_image_PreviousPlaques_96h” ,“ Output_image_OverlayPlaques_72queh”和“ Output_image_lay”)。

斑块追踪管道


1.图像:只需拖放噬斑测定荧光图像(例如,“ Input_image_mNeptune_72h”)文件和使用先前噬斑识别管线创建的输出图像(例如,“ Output_image_PreviousPlaques_72h”)文件即可。确保空斑测定荧光图像做的名称包括其中被测量的荧光的信道(的名称例如,mNeptune )和时间推移显微镜图像增加连续编号(例如,mNeptune_72h)。     

2. NamesAndTypes :硒LECT用表达规则标准包含在所有的噬斑测定荧光图像的名称,在这种情况下,指示的荧光的信道(例如,mNeptune )。对于每个荧光图像,它将创建一个称为“传感器”的图像。     

3.模块3-8与“斑块识别”管道中描述的模块相同。     

4. IdentifySecondaryObjects :此模块将“ CellImage ”中的新Plaque对象标识为先前标识的PreviousPlaques主要对象周围的次要对象。     

5. TrackObjects :此模块使用时间分辨图像上的重叠标准随时间跟踪斑块,并创建名为“ TrackedPlaques ”的输出图像。您可以调整“最大距离”以考虑匹配,以实现最佳噬菌斑追踪。     

6. SaveImages :保存“ Tracked_Plaques ”图像以供您验证和最终结果。     

7. RelateObjects :此模块将新的Plaque对象与先前标识的Nuclei对象相关联。     

8. ExporttoSpreadSheet :此模块将构成所有已分析图像中每个可识别斑块的细胞数(核对象)的数据导出到excel电子表格。     

注意:与该协议一起,我们提供Zip文件“ PlaqueIdentification跟踪管道和数据集”,其中包含应用的管道(“ PlaqueTracking.cpproj ”)和一个示例数据集,该示例数据集由运行管道的四个输入图像组成(“ Input_image_mNeptune_72h”,“ Input_image_mNeptune_96h” ”,“ Output_image_Previous Plaques_72h”和“ Output_image_PreviousPlaques_96h”)以及三个输出文件来确认预期结果(“ Tracked_Plaques_72h”,“ Tracked_Plaques_96h”和“ Viral_Plaque ”)。

菜谱

1.完整的DMEM      


500毫升DMEM(Gibco )


5毫升抗生素-抗真菌药100 ×


5毫升(2%)或50毫升(10%)的FBS


用手旋转匀浆(20次,轻轻地避免形成泡沫)


储存在4℃。稳定4个月


2. PEI溶液(1毫克/毫升)      


1毫克PEI粉


1 ml蒸馏水中的0.2 M HCl


热和摇,在60℃,600rpm下在热混合器®块


过滤灭菌(孔径0.2 µm)


将45 µl等分试样准备入2 ml反应管中,并在-80°C下保存。稳定4个月


3.聚乙烯溶液(10毫克/毫升)      


10.6毫克六溴二苯醚粉末


溶解在1毫升的destilled水


过滤灭菌(孔径0.2 µm)


将50 µl单次使用的等分试样准备到0.5 ml反应管中,并储存在-20°C下。稳定1年


4.完成MEM      


500 ml MEM(Gibco )


5 ml丙酮酸钠100 mM


5毫升抗生素-抗真菌药100 ×


5毫升(2%)或50毫升(10%)的FBS


用手旋转匀浆(20次,轻轻地避免形成泡沫)


储存在4℃。稳定4个月


5. PBS 1%FBS      


45 ml PBS,pH 7.4


5毫升完整MEM 10%FBS


储存在4℃。稳定4个月


6.嘌呤霉素溶液(10 mg / ml)      


10毫克嘌呤霉素


溶解在1毫升的destilled水


过滤灭菌(孔径0.2 µm)


将20 µl单次使用的等分试样准备到1.5 ml的光保护反应管中,并储存在-20°C下。稳定2年


7. FluoroBrite TM DMEM 2%FBS      


500毫升FluoroBrite TM DMEM(Gibco )


5毫升GlutaMAX TM补品


5 ml丙酮酸钠100 mM


5毫升抗生素-抗真菌药100 ×


5毫升的胎牛血清


用手旋转匀浆(20次,轻轻地避免形成泡沫)


储存在4℃。稳定4个月


8.斑块培养基2%FBS      


9.4克MEM(Sigma)


900毫升Milli -Q水


调节pH至4.0


10克羧甲基纤维素钠盐


高压灭菌(121°C在100 kPa下15分钟)


在Milli- Q水中的30 ml 7.5%碳酸氢钠溶液(通过过滤灭菌-孔径0.2 µm)


10毫升GlutaMAX TM补品


10 ml丙酮酸钠100mM


10毫升抗生素-抗真菌药100 ×


20毫升FBS


用手旋转匀浆(20次,轻轻地避免形成泡沫)


储存在4℃。稳定4个月

致谢

我们要感谢马萨诸塞大学化学系的Jeanne A. Hardy博士在我们出色的合作过程中所表现出的友善和科学建议。我们还要感谢哥斯达黎加大学(项目VI-803-B9-505)和国际基因工程与生物技术中心(GRP CRP / CRI18-02)的资助来源。 

利益争夺

作者宣称他们没有任何利益冲突。

参考

阿里亚斯·阿里亚斯(Arias-Arias),JL,麦克弗森(MacPherson),DJ,希尔(Hill),缅因州(Me),哈迪(Hardy),JA和莫拉·罗德里格斯(Mora-Rodriguez)R.(2020年)。黄病毒NS2B-NS3蛋白酶活性的可激活荧光的报告基因使单细胞和病毒斑块中感染的实时成像成为可能。Ĵ生物学化学295(8):2212至2226年。
              Balsitis ,SJ,Coloma,J.,Castro,G.,Alava,A.,Flores,D.,McKerrow ,JH,Beatty,PR和Harris,E。(2009)。登革热病毒在小鼠和人类中的流行性,是由病毒非结构蛋白3特异性免疫染色确定的。Am J Trop Med Hyg 80(3):416-424 。
Freppel ,W.,Mazeaud ,C.和Chatel-Chaix ,L.(2018)。寨卡病毒在哺乳动物细胞中的产生,滴定和成像。生物协议8(24):e3115。
              古尔德(EA)和所罗门(T.)(2008)。致病性黄病毒。柳叶刀371(9611):500-509。
Hawley,TS,Herbert,DJ,Eaker ,SS和Hawley,RG(2004)。荧光蛋白报告基因的多参数流式细胞术。方法分子生物学263:219-237。
谢女士(MS,Chen,MY),谢女士(CH),潘卓(CH),余桂英(Y,GY)和陈洪(2017)。使用基于登革热NS3蛋白酶活性的新型生物传感器系统检测和定量登革热病毒。PLoS One 12(11):e0188170。
ķ ü mmerer ,BM(2018)。黄病毒复制子的建立和应用。Adv Exp Med Biol 1062:165-173。
Li SH,Li XF,Zhao,H.,Deng,YQ,Yu,XD,Zhu,SY,Jiang,T.,Ye,Q.,Qin,ED and Qin,CF(2013)。日本脑炎活疫苗病毒SA14-14-2的复制子系统的开发和表征。维罗尔J 10:64 。
              McFadden,MJ,Mitchell-Dick,A.,Vazquez,C.,Roder ,AE,Labagnara ,KF,McMahon,JJ,Silver,DL和Horner,SM(2018)。一个基于荧光细胞的系统,用于实时成像寨卡病毒感染。病毒10(2)。              
Medin ,CL,Valois,S.,Patkar ,CG和Rothman,AL(2015)。基于质粒的报告系统,用于登革热病毒感染细胞的活细胞成像。J Virol方法211:55-62。
麦地那(F.),麦地那(F),麦地那(JF),科隆(C.),韦尔涅(E.),佐治亚州圣地亚哥(Santiago)和佐治亚州Munoz-Jordan(2012)。登革热病毒:隔离,繁殖,定量和储存。Curr Protoc Microbiol第15章:15D单元12。
              施密德,B.,里纳斯,M.,罗明坚,A.,科斯塔,EG,Bartenschlager ,M.,路透,A.,Fischl ,W.,哈德,N.,Bergeest ,JP Flossdorf ,M.,罗尔, K.,ħ ö FER ,T。和Bartenschlager ,R。(2015)。活细胞分析和数学建模确定了登革热病毒2'-o-甲基化突变体减毒的决定因素。PLoS Pathog 11(12):e1005345。
              Soto-Garita,C.,Somogyi,T.,Vicente-Santos,A.和Corrales-Aguilar,E.(2016)。哥斯达黎加两次登革热暴发的分子特征。Am J Trop Med Hyg 95(1):201-205 。              
田村,福冈,内田,小野,森,H.,佐藤,A.Fauzyah ,Y.,冈本,T.,Kurosu ,T.,濑户,YX,今村,M.,Tautz ,N.,Sakoda ,Y.,Khromykh ,AA,Chayama ,K.和Matsuura,Y.(2018)。具有小的报告子标签的重组黄病毒科病毒的表征。J Virol 92(2)。
Tiscornia ,G.,Singer,O。和Verma ,IM(2006)。慢病毒载体的生产和纯化。纳特Protoc 1(1):241-245。
谢X.,邹J.,山C.,杨Y.,库姆DB,达米尔,K.,Neyts J. Zika病毒复制子,用于药物发现。EBioMedicine 12:156-160。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Arias-Arias, J. L. and Mora-Rodríguez, R. (2021). Generation and Implementation of Reporter BHK-21 Cells for Live Imaging of Flavivirus Infection. Bio-protocol 11(5): e3942. DOI: 10.21769/BioProtoc.3942.
  2. Arias-Arias, J. L., MacPherson, D. J., Hill, M. E., Hardy, J. A. and Mora-Rodriguez, R. (2020). A fluorescence-activatable reporter of flavivirus NS2B-NS3 protease activity enables live imaging of infection in single cells and viral plaques.J Biol Chem 295(8): 2212-2226.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。