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Jun 2018

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Method for Studying the Effect of Gene Silencing on Bacterial Infection-induced ERK1/2 Signaling in Bone-marrow Derived Macrophages
基因沉默对于细菌感染诱导骨髓巨噬细胞中ERK1/2信号通路的影响研究方法   

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Abstract

Macrophages are highly phagocytic cells that utilize various pathogen recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). These PAMPs can be present within the microbe, such as bacterial CpG DNA, and are recognized by Toll-like receptor 9 (TLR9), a PRR present on the endosomal membrane of macrophages. PAMPs can also be present on the surface of microbes, such as Lipopolysaccharide (LPS), which decorates the outer membrane of gram-negative bacteria like Salmonella typhimurium and Escherichia coli. LPS is recognized by TLR4 present on the plasma membrane of macrophages, and LPS-TLR4 association leads to activation of signaling cascades including MAPK phosphorylation, which in turn promotes macrophage activation and microbial killing. This protocol describes the method for studying the role of a gene of interest in Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling, induced by bacterial infection in primary bone-marrow derived macrophages (BMDMs).

Keywords: Bone-marrow derived macrophages (骨髓巨噬细胞), Salmonella (沙门氏菌), Infection (感染), ERK signaling (ERK信号通路), Western blotting (蛋白质印迹)

Background

Macrophages are phagocytic cells which can either be resident to specific tissues as Kupffer cells (in the liver) and peritoneal macrophages (in peritoneal cavity) or, can enter tissues in response to an infection. The primary function of macrophages involves phagocytosis and clearance of old damaged cells, and recycling of nutrients in the serum (recently reviewed in Shapouri-Moghaddam et al., 2018). However, for macrophages to clear microbes, there exists a need for their activation. Macrophages obtained from many tissues such as alveoli and peritoneal cavity includes high numbers of pre-activated macrophage population. However, macrophages derived from myeloid progenitor cells present in the bone marrow are comparatively naive and more responsive to activating stimulus (Epelman et al., 2014). Upon encounter of gram-negative bacteria by macrophages, LPS present on either the surface of a bacterium or shed by bacteria in the blood flow is captured by LBP (LPS-binding protein) and presented as a ligand to TLR4, a type I transmembrane protein present on the plasma membrane of macrophage that mediate the recognition of PAMPS such as LPS (Shimazu et al., 1999). The engagement of LPS with TLR4 (along with other co-stimulatory molecules) leads to recruitment of several adaptor proteins at the cytoplasmic tail of TLR4 followed by a cascade of intracellular events leading to activation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling cascades downstream to TLR4 including ERK1/2 (Buscher et al., 1995; Schaeffer and Weber, 1999; Gay et al., 2014; Platko et al., 2018; Arya et al., 2018). These signaling pathways directly or indirectly phosphorylate and activate various transcription factors that lead to the expression of genes involved in production and release of pro-inflammatory cytokines, reactive oxygen species, nitrosative burst and promotes macrophage activation (Satoh and Akira, 2016). Thus, ERK1/2 signaling plays an important role in augmenting macrophage response against intracellular pathogens. This protocol describes a method to investigate the role of a host factor of interest in modulating ERK1/2 activation in primary BMDMs in response to Salmonella typhimurium infection (see schematic shown in Figure 1 and Arya et al., 2018).

Materials and Reagents

  1. 5 ml syringe (Dispovan)
  2. 60-mm tissue culture dish (BD Falcon®, catalog number: 353002)
  3. 10-cm tissue culture dish (BD Falcon®, catalog number: 353003)
  4. 6-well tissue culture plate (BD Falcon®, catalog number: 353046) 
  5. 50 ml Falcon tube (BD Falcon®, catalog number: 352070)
  6. 15 ml Falcon tube (BD Falcon®, catalog number: 352096)
  7. 14 ml round-bottom polypropylene tube (Corning, catalog number: 352006)
  8. 1.5 ml microcentrifuge tube (MCT) (Tarson, catalog number: 50010)
  9. 0.2 micron PVDF membrane (Bio-Rad, catalog number: 162-077)
  10. X-ray film (Carestream, catalog number: 6574958)
  11. Aluminum foil
  12. Cell scraper (HiMedia, catalog number: TCP104)
  13. Sterile 22 G needle (Dispovan)
  14. C57BL/6 male mice (typically 6-8 weeks old)
  15. Salmonella typhimurium strain SL1344
  16. RPMI medium 1640 (Lonza, catalog number: 12702F)
  17. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147)
  18. HEPES (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080)
  19. MEM Non-Essential Amino Acids Solution (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 11140-050)
  20. Antibiotic-Antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240-062)
  21. Sodium Pyruvate (100 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11360-070)
  22. GlutaMAXTM Supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 35050-061)
  23. DPBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-144) 
  24. PBS, pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023) 
  25. Trypsin-EDTA (0.05%) (Thermo Fisher Scientific, GibcoTM, catalog number: 25300-054) 
  26. Trypan Blue Solution (Thermo Fisher Scientific, GibcoTM, catalog number: 15250-061) 
  27. Mouse M-CSF (macrophage-colony stimulating factor) (eBiosciences, catalog number: 34-8983)
  28. ACK (Ammonium-Chloride-Potassium) lysing buffer (Thermo Fisher Scientific, GibcoTM, catalog number: A10492-01)
  29. Protease inhibitor (Sigma-Aldrich, catalog number: P8340)
  30. Phosphatase inhibitor (Roche, catalog number: 4906837001)
  31. Bradford reagent (Sigma-Aldrich, catalog number: B6916)
  32. TWEEN® 20 (Sigma-Aldrich, catalog number: P9146) 
  33. TritonTM X-100 (Sigma-Aldrich, catalog number: T8787) 
  34. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771) 
  35. Sodium deoxycholate (DOC) (Sigma-Aldrich, catalog number: D6750)
  36. Trizma® base (Tris base) (Sigma-Aldrich, catalog number: T6066) 
  37. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 3014)
  38. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: 3889)
  39. Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA-Na2) (Sigma-Aldrich, catalog number: E5134)
  40. Gentamicin solution (Sigma-Aldrich, catalog number: G1272)
  41. Streptomycin sulfate (Sigma-Aldrich, catalog number: S6501)
  42. Ethanol (Merck, catalog number: 108543)
  43. Skim milk (BD Difco, catalog number: 232100)
  44. LB broth (BD Difco, catalog number: 244620)
  45. SS agar (HiMedia, catalog number: M108) 
  46. Laemmlisample buffer (Bio-Rad, catalog number: 161-0747)
  47. ON-TARGETplus Non-targeting Control siRNA Pool (Dharmacon, catalog number: D-001810-10) 
  48. ON-TARGETplus Mouse Arl11 siRNA (Dharmacon, catalog number: L-055672-01-0005)
  49. Polyclonal anti-total ERK1/2 antibody (Cell Signaling Technology, catalog number: 4695)
  50. Polyclonal anti-phospho-ERK1/2 antibody (Cell Signaling Technology, catalog number: 4370)
  51. Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, catalog number: 111-035-144)
  52. ECL Plus Western Blotting Substrate (Pierce, catalog number: 32132)
  53. 70% ethanol (see Recipes)
  54. Complete RPMI medium (see Recipes)
  55. Mouse M-CSF stock solution (0.4 mg/ml) (see Recipes)
  56. SS agar plate (see Recipes)
  57. Streptomycin stock solution (50 mg/ml) (see Recipes)
  58. LB broth (see Recipes)
  59. Opsonization buffer (see Recipes)
  60. Growth medium (GM) for infection (see Recipes)
  61. RIPA lysis buffer (see Recipes)
  62. Blocking buffer (see Recipes)
  63. PBST (see Recipes)
  64. Antibody dilution buffer (see Recipes)

Equipment

  1. Beaker
  2. 0.2 micron polyethersulfone filter
  3. Scissors and forceps (Fisher Scientific)
  4. Cell culture CO2 incubator (Thermo Scientific, model: 371)
  5. Biosafety cabinet class II (ESCO, model: AC2-4S8-NS)
  6. Inverted tissue culture microscope (Nikon, model: TS2) 
  7. Hemocytometer (Sigma, catalog number: Z359629)
  8. Multimode reader (Tecan, model: Infinite M200)
  9. Incubator shaker (New Brunswick, model: Innova 42) 
  10. Automatic X-ray Film Processor (Protec, model: OPTIMAX 2010)
  11. Flatbed scanner (Microtek, model: Bio-5000)
  12. Refrigerated centrifuge with plate centrifuge bucket big enough for a 6-well plate (Eppendorf, model: 5810 R)
  13. Semi-dry horizontal blotting system (Atto, model: WSE-4020)
  14. SDS-PAGE gel apparatus (Bio-Rad, model: Mini Protean Tetra Cell)
  15. Dancing shaker (Tarson, model: MC-02)
  16. Rocking shaker (Tarson, model: 4080)
  17. Flow cytometry

Software

  1. ImageJ software (NIH, https://imagej.nih.gov/ij/)
  2. Microsoft Excel software (MS Office)

Procedure

This protocol is divided into three parts: A) Generation of mouse primary BMDMs; B) Infection of BMDMs with Salmonella typhimurium; and C) Assessment of activated/phosphorylated levels ERK1/2 in Salmonella-infected macrophage cell lysates by Western Blotting. The readers are encouraged to see the schematic diagram shown in Figure 1 that describes the different steps for carrying out this protocol.


Figure 1. Schematic diagram showing the steps used to investigate the role of a host factor of interest in modulating ERK1/2 activation in primary bone marrow-derived macrophages (BMDMs) in response to Salmonella typhimurium infection. Isolate mouse bone marrow cells and culture in the presence of M-CSF to generate primary BMDMs. After generation of BMDMs transfect the cells with either control siRNA or siRNA against the gene of interest. Post-siRNA treatment, infect the BMDMs with opsonized stationary phase culture of Salmonella typhimurium. At the end of different times post infection, perform cell lysis and run the lysates on SDS-PAGE gel, perform the Western Blotting and develop the signal on X-ray films using chemiluminescent substrate. Quantify the signal intensity from the scanned X-ray films using ImageJ software.


  1. Generation of mouse primary BMDMs
    1. Prepare for mouse dissection by keeping three 60-mm tissue culture dishes filled with 70% ethanol (see Recipe 1), DPBS or complete RPMI media (see Recipe 2), and a pair of sterile (autoclaved) forceps and scissors over the clean area inside the biosafety cabinet (BSC). Also, place a square-shaped (12 x 12 inches) aluminum foil cleaned with 70% ethanol to be used as a platform for mouse dissection.
      Note: Sterile conditions to be maintained all the time during the experiment.
    2. Sacrifice mouse by cervical dislocation and immerse mouse in a beaker containing 70% ethanol. Using the tip of a small scissor make a cut near the thigh of the mouse and peel off the skin from the top of each hind limb down over the foot using scissors and forceps. Detach hind limb from the body by cutting the caudal bone along with the femur (cut along the bone that feels 90° to the femur). Cut off the foot (tarsus) along with the skin and discard. Keep the de-skinned limbs in the dish containing 70% ethanol for 5 min as this will make muscles dehydrate making them easier to detach from bone (see Figures 2A-2G).
      Note: Remove muscles as aim of this step is to prevent contamination of BMDM cultures with muscle and fibroblast cells.
    3. Remove excess muscles from the limbs using scissors while maintaining bone integrity. Detach the knee joint carefully by pressing softly with scissors to cut-off through the ligaments between femur and tibia (see Figures 2H-2I). Keep bones in 70% ethanol for 5 min and then transfer them to the dish containing DPBS for 5 min.
    4. Hold the bone at the center using a pair of forceps and cut both the ends (~3-6 mm from the end) of the tibia/femur using a scissor. Fill-up the 5 ml syringe with complete RPMI media and flush the bone marrow (BM) through one end of the bone into the 60-mm tissue culture dish containing complete RPMI media. Repeat this step through the other end of the bone to expel the BM from both ends of the bone (Figures 2J-2K).
    5. Using a 5 ml syringe, pass the collected BM through a 22 G needle 3-4 times to make single cell suspension and collect cells in a 50 ml Falcon tube (Figures 2L-2N).
    6. Centrifuge the cell suspension at 700 x g for 5 min at room temperature (RT), and decant the supernatant.
    7. Add 1 ml ACK lysing buffer to the cell pellet to lyse the RBCs and continue tap mixing for an incubation period of 1 min. Immediately add 10 ml DPBS and centrifuge the cell suspension at 700 x g for 5 min at RT (Figure 2O).
      Note: Do not exceed the incubation time beyond 1 min as it might cause damage to cells other than RBC, and decrease overall yield of BMDMs.
    8. Wash the cell pellet two times using 10 ml DPBS as described in Step A6.
      Note: While adding DPBS observe for muscle contamination. If any muscle debris is present, take it out by using 1 ml pipette.
    9. Following the last wash, resuspend the cell pellet in 15-20 ml of complete RPMI media and count the cells with a hemocytometer under an inverted tissue culture microscope using the trypan blue exclusion principle.
      Note: The yield of BM cells varies a lot and accordingly different dilution of trypan blue should be used to get accurate cell count. Typically, one can use a starting dilution of 1:100 of trypan blue with an incubation period of 1 min for cell counting.
    10. Seed ~4 million BM cells in a 10-cm tissue culture dish containing 8 ml of complete RMPI media supplemented with 30 ng/ml of M-CSF (see Recipe 3). Place the dishes in a cell culture incubator at 37 °C and 5% CO2. Consider this step as Day 1.
    11. Observe for the increase in the percentage of adhered macrophages on Day 2.
    12. On Day 3, replenish the BM culture with fresh media by adding half the volume of complete RPMI media that was initially used, i.e., 4 ml complete RPMI media supplemented with M-CSF (30 ng/ml). 
    13. Observe for the increase in the percentage of adhered macrophages (around 60%) on Day 4.
    14. By Day 5, ~90% adherent macrophages will be observed (Figure 3). To use BMDMs in functional experiments, remove the media, and gently wash the cells with 10 ml DPBS. To detach the BMDMs, add 5 ml trypsin and place the dish inside the incubator for 5-10 min at 37 °C. After the incubation period, take a quick look at the cells under the microscope to confirm detachment. Next, perform trypsin neutralization by adding 5 ml complete RPMI media. Using a 1 ml pipette tip gently flush the cells and transfer the cell suspension into a 50 ml centrifuge tube.
      Note: Perform step A14 very gently to avoid activation of macrophages.
    15. Centrifuge the cell suspension at 700 x g for 5 min at RT, gently resuspend in 10 ml complete RPMI media and perform cell counting using a hemocytometer as described in Step A9.
    16. At this step, the collected cells can be analyzed for macrophage purity by flow cytometry and are ready for further characterization and functional experiments.
      Note: For analyzing the purity of isolated macrophages by flow cytometry, readers can refer to Zhang et al., 2008.


      Figure 2. Dissection of mouse for the preparation of BMDMs. A. A 6-8 week-old C57BL/6 male mouse was sacrificed by cervical dislocation and sterilized in 70% ethanol and placed on a clean aluminum foil inside the biosafety cabinet. B-E. Using a small scissor make a cut near the thigh of the mouse and peel off the skin from the top of each hind limb down over the foot using scissors and forceps. F. Detach hind limb from the body by cutting the caudal bone along with the femur. G. Cut off the foot (tarsus) along with the skin. H and I. Remove excess muscles from the limbs using a scissor and detach the knee joint carefully by pressing softly with a scissor to cut-off through the ligaments between femur and tibia. J and K. Hold the bone at the center using forceps and cut both the ends of the tibia/femur using a scissor. L-N. Using a 5 ml syringe, pass the collected BM through a 22 G needle 3-4 times to make single cell suspension and collect cells in a 50 ml Falcon tube. O. Centrifuge and add ACK lysing buffer to the cell pellet to lyse the RBCs. Immediately DPBS and centrifuge the cell suspension to collect BM cells.


      Figure 3. Characterization of mouse BMDMs. BM cells isolated from mouse hind limbs were cultured in the presence of M-CSF. On Day 5, the macrophage population was largely adherent. Scale bar = 100 µm.

  2. Infection of BMDMs with Salmonella typhimurium
    1. Seed BMDMs at a density of 1 x 106 cells/well in a 6-well tissue culture plate in 2 ml complete RPMI media containing 30 ng/ml per well of mouse M-CSF. The number of 6-well tissue culture plates required will depend upon the number of siRNA treatments to be performed and different time points of infection to be analyzed. 
    2. Next day, replace the media with fresh complete RPMI media containing 30 ng/ml of mouse M-CSF and transfect BMDMs with control siRNA and gene specific siRNA as per the manufacturer’s instructions. To illustrate this protocol, we have performed control siRNA or Arl11 siRNA as described in Arya et al., 2018.
      Note: Readers are encouraged to standardize the conditions i.e., the amount of siRNA and treatment time in order to achieve the maximum silencing for the gene of their interest. 
    3. To prepare stationary culture for infection, inoculate an isolated colony of Salmonella typhimurium SL1344 strain from a freshly streaked SS agar plate containing 50 μg/ml streptomycin (see Recipes 4 and 5) in 3 ml LB broth (see Recipe 6) supplemented with streptomycin (50 μg/ml) in a 14 ml round-bottom polypropylene tube. Set up an overnight culture (~16 h) in a shaking incubator (200 rpm) at 37 °C. This step can be initiated at 56 h post siRNA treatment of BMDMs so that the duration of siRNA treatment (72 h) is completed by the time Salmonella culture is ready for infection.
      Note: Salmonella typhimurium SL1344 strain is streptomycin resistant and streptomycin is added in the culture to prevent growth of any other contaminants.
    4. Measure O.D. of overnight Salmonella culture (diluted to 1/10 in LB broth) at 600 nm wavelength using a spectrophotometer. Transfer the volume of bacterial culture equivalent to an O.D. = 1 (~109 live bacteria) in a MCT. Check the example calculation to estimate the volume required for an O.D. = 1.
      Example calculation:
      If O.D.600 of 1/10 dilution of overnight bacterial culture = 0.33
      Thus, calculated O.D.600 of bacteria in 1 ml culture will be = 0.33 x 10 = 3.3
      Number of bacteria equal 10in 1 ml, i.e., O.D.600 = 1.0
      Therefore, volume of bacterial culture required = (1/Actual O.D.600) x 1 ml= (1/3.3) x 1 ml = 0.303 ml
      Transfer 0.303 ml of bacterial culture to a fresh MCT and make up the volume to 1 ml by adding 0.697 ml LB broth.
    5. Centrifuge the diluted Salmonella culture at 8,600 x g for 3 min at RT, aspirate 0.9 ml LB broth and add equivalent volume of DPBS. Resuspend the bacterial cells by gentle vortexing and repeat the centrifugation step again to remove the LB broth.
      Note: After centrifugation out of 1 ml only 0.9 ml volume is aspirated to prevent any loss of bacteria, and the centrifugation step is repeated twice in order to remove the LB broth completely.
    6. Perform opsonization of bacteria. For efficient adherence of bacteria on the macrophage surface and phagocytosis, the bacterial surface is coated with IgG present in serum. Aspirate 0.9 ml DPBS after completion of Step B5 and resuspend the bacterial pellet in 900 μl opsonization buffer (see Recipe 7). Incubate the bacterial suspension at 37 °C for 20 min with intermittent invert mixing, followed by centrifugation at 8,600 x g for 3 min. Gently aspirate the supernatant (~0.9 ml) and add 0.9 ml DPBS to resuspend the bacterial cell pellet.
      Note: After centrifugation out of 1 ml only 0.9 ml volume is aspirated to prevent any loss of opsonized bacteria.
    7. Dilute the opsonized bacteria to 1/10 in GM (see Recipe 8), i.e., 4.5 ml GM (pre-warmed to 37 °C) + 0.5 ml of the opsonized bacteria. This implies, if O.D.600 = 1 means 1 x 109 bacteria/ml then 1/10 dilution will have 108 bacteria/ml.
      Note: The Steps B6 and B7 should be performed in separate biosafety cabinets in order to minimize contamination.
    8. Infect the plated BMDMs from Step B2 (i.e., post 72 h siRNA treatment) at a multiplicity of infection (MOI) 50:1. To perform this, remove the complete RPMI media and wash once with DPBS. Then quickly add 0.5 ml of diluted bacteria from Step B7 to each well. MOI is the number of bacteria added per cell during infection. See the example calculation to estimate the volume required to achieve an MOI of 50:1.
      Example calculation:
      BMDMs plated in a 6-well tissue culture dish at a density of 106 cells per well
      So, for MOI = 50:1, number of bacteria required = 106 x 50 = 5 x 107
      We have 108 bacteria in 1 ml (as in Step B7)
      Therefore for 5 x 107, culture volume required = 1 ml x 5 x 107/108 = 0.5 ml bacterial dilution per well.
    9. To avoid non-uniform phagocytosis of Salmonella by BMDMs synchronize the process by adding 1.5 ml GM to each well of a 6-well tissue culture plate, seal the plate with Parafilm to avoid spillage, and perform a centrifuge at 453 x g at 30 °C
      for 5 min.
    10. Remove the Parafilm and transfer the plate to the cell culture incubator at 37 °C and 5% CO2 for 20 min post infection (p.i.) sample.

  3. Assessment of activated/phosphorylated levels ERK1/2 in Salmonella-infected macrophage lysates by Western Blotting
    1. Aspirate the media from the wells to remove non-internalized bacteria and give one wash with DPBS (1 ml/well) and remove the DPBS completely without dislodging the cells. 
    2. Preparation of cell lysates
      Lyse the uninfected (control) and 20 min p.i. samples by adding ice-cold RIPA buffer supplemented with 1x protease inhibitors and 1x phosphatase inhibitors (0.1 ml per well of 6-well plate) (see Recipe 9). Immediately scrape the cells off the plate and transfer the extract to an MCT properly labeled with sample name. Keep on ice.
    3. To the remaining sample wells, add 2 ml of GM (supplemented with 50 μg/ml gentamicin), and transfer the plate to a tissue culture incubator at 37 °C and 5% CO2 for the next time interval of infection as per your experiment requirement. For example, for the 50 min p.i. sample, incubate the sample well for 30 min. At the end of each time p.i., prepare the cell lysates as described in Step C2.
      Note: Gentamicin is added to the media in order to prevent the growth of non-internalized bacteria which are left over even after washing, and also gentamicin exhibit minimal cytotoxicity to macrophages.
    4. Keep the cell lysates on ice for up to 10 min (after scraping), followed by centrifugation at 16,600 x g at 4 °C for 10 min.
    5. Carefully collect the post nuclear supernatant (PNS) in a fresh MCT without disturbing the pellet. The PNS can be stored at -80 °C if SDS-PAGE is to be performed next day.
    6. Quantify the concentration of protein present in the PNS by Bradford assay using the manufacturer’s instructions. Normalize all the samples with respect to each other in order to load equal amount of protein (~15-20 μg per lane).
    7. After normalization calculate the volume required of each sample and transfer to fresh MCT and add Laemmli sample buffer to a final concentration of 1x, boil at 99 °C for 7 min followed by a short spin.
    8. Load samples onto 10% SDS-PAGE gels and follow the standard Western Blotting procedure.
      Note: The SDS-PAGE gels need to be run in duplicate since antibodies against p-ERK1/2 and total-ERK1/2 detect band at the same size.
    9. After performing the protein transfer step (30 Volts for 2 h in cold room), incubate PVDF membrane in blocking buffer (see Recipe 10) for overnight at 4 °C on a horizontal shaker.
    10. Remove the blocking buffer and wash the membrane three times for 5 min each with 0.05% PBST (see Recipe 11). 
    11. Incubate membrane and primary antibody (at 1:1,000 dilution) in the antibody dilution buffer (0.05% PBST; see Recipe 12) with gentle agitation for 2 h at RT.
    12. Wash the membrane three times for 5 min each with 0.05% PBST on a dancing shaker.
    13. Incubate membrane with goat anti-rabbit IgG (HRP-conjugated) antibody (1:5,000 dilution) in 0.05% PBST with gentle agitation for 1 h at RT.
    14. Remove the secondary antibody solution and wash the membrane three times for 10 min each with 0.3% PBST on a dancing shaker.
    15. Prepare the ECL reagent as per the manufacturer’s instructions. Incubate the ECL substrate with membrane for 1 min, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film and develop the signal in Automatic X-ray film processor (Figure 4).
    16. Scan the developed X-ray films using the flatbed scanner and perform the densiotometric quantification of phospho- and total-ERK1/2 band signal using ImageJ software as described in the data analysis section below.


      Figure 4. Arl11 depletion in BMDMs impairs ERK1/2 activation upon infection with Salmonella. Control- and Arl11-silenced BMDMs were infected with Salmonella typhimurium for different time periods, and lysates were prepared and blotted with the indicated anti-phospho-ERK1/2 and anti-total-ERK1/2 antibodies.

Data analysis

Densitometric quantification of phospho- and total-ERK1/2 signal using ImageJ software

  1. Launch ImageJ software and open the scanned images of phospho-ERK1/2 and total-ERK1/2 blots as shown in Figure 5A. Go to “File” → “Open” → “Browse” → “Select File” → “Open”.
  2. Select the “Rectangle Tool” from the ImageJ toolbar and draw a box which covers the band signal in lane 1 as shown in Figure 5B. Go to “Analyze” → “Gels” → “Select First Lane”.
  3. Drag and drop the selection from lane 1 to lane 2 as shown in Figure 5C. Then go to “Analyze” → “Gels” → “Select Next Lane”. Repeat this step until all the lanes are selected.
  4. To get the pixel intensity of each band, go to “Analyze” → “Gels” → “Plot Lanes”. Then select the “Wand tool” from the Image J toolbar and place the cursor (+) at the center of histogram for lane 1 and repeat this step for the next lanes and left click with the mouse to obtain area under the peak (pixel intensity) in a separate box as shown in Figure 5D. To perform this step, make sure “Area Dialog Box” is checked. To check for this, go to “Analyze” → “Set Measurements” → “Check Area Dialog Box” → Hit “OK”.
  5. Copy the measurements to MS Excel Sheet for both phospho-ERK1/2 and total-ERK1/2 blots for each time point of infection. Then calculate the ratio of phospho-ERK1/2 to total-ERK1/2 signal for each time p.i. and plot a clustered column graph. For more information, readers are encouraged to refer Figures 4a and 4b in Arya et al., 2018.


    Figure 5. Densitometric quantification using ImageJ. Screenshots of different steps of densitometric quantification using ImageJ software are shown. For details refer to data analysis section of the protocol.

Recipes

  1. 70% ethanol
    Mix 70 ml of absolute ethanol with 30 ml of sterile distilled water (DW)
  2. Complete RPMI medium
    RPMI 1640
    10% Heat-inactivated FBS
    10 mM HEPES
    1 mM Sodium pyruvate
    1x Antibiotic-Antimycotic
    1x MEM Non-essential Amino acids
    1x GlutaMAXTM Supplement
    Store at 4 °C
  3. Mouse M-CSF stock solution (0.4 mg/ml)
    0.5 mg M-CSF
    1.25 ml PBS (pH 7.4)
    Filter sterilize using 0.2-micron polyethersulfone filter, aliquot and store at -80 °C
    Note: To increase the shelf-life (upto1 year), use 0.1% BSA in PBS (pH 7.4) as solvent.
  4. SS agar plate
    6.3 g
    100 ml DW
    Heat to boiling with frequent agitation to dissolve the medium completely. Cool to about 45 °C, add the required concentration of antibiotic, mix well and pour into sterile Petri plates. Store the plates at 4 °C
    Note: Do not autoclave or overheat SS-Agar media as it will destroy the selectivity of the medium. 
  5. Streptomycin stock solution (50 mg/ml)
    0.5 g Streptomycin sulfate
    10 ml DW
    Filter sterilize, aliquot and store at -80 °C
  6. LB broth
    2.5 g LB mix
    100 ml DW
    Autoclave and store at RT in sterile conditions
  7. Opsonization buffer
    20% FBS in DPBS
  8. GM for infection
    RPMI 1640
    10% Heat-inactivated FBS
    10 mM HEPES
    1 mM Sodium pyruvate
    1x MEM Non-essential Amino acids
    1x GlutaMAXTM Supplement
    Store at 4 °C
    Note: In the infection media no antibiotic is added in order to prevent arrest and killing of internalized bacteria.
  9. RIPA lysis buffer
    10 mM Tris-Cl (pH 8)
    140 mM NaCl
    1 mM EDTA
    0.5 mM EGTA
    1% TritonTM X-100
    0.1% DOC
    0.1% SDS
    Store at RT
    Note: Add protease and phosphatase inhibitors to RIPA lysis buffer before immediate use.
  10. Blocking buffer
    5% w/v non-fat dry milk in 0.05% PBST
  11. PBST
    Required percentage of TWEEN® 20 in PBS
    Mix well by stirring and store at RT
  12. Antibody dilution buffer
    Primary and secondary antibodies dilution (as per the antibody datasheet) made in 0.05% PBST

Acknowledgments

This protocol was adapted and modified from previously published studies (Sindhwani et al., 2017; Arya et al., 2018). The authors acknowledge financial support from Council of Scientific & Industrial Research (CSIR), India. A. Tuli acknowledges financial support from the Wellcome Trust/Department of Biotechnology (DBT). This work was supported by the Wellcome Trust/DBT India Alliance Intermediate Fellowship awarded to A. Tuli (IA/I/14/2/501543). A. Tuli also acknowledges the infrastructure and financial support from CSIR-Institute of Microbial Technology (IMTECH, Chandigarh) (OLP-144, communication No. 038/2018).

Competing interests

The authors declare no competing financial interests.

Ethics

This study was carried out in strict accordance with the guidelines issued by the Committee for the Purpose of Supervision of Experiments on Animals (1094 55/1999/CPCSEA) under the Prevention of Cruelty to Animals Act 1960 and amendments introduced in 1982 by the Ministry of Environment and Forest, Government of India. All protocols involving mouse experiments were approved by the institutional animal ethics committee of the Council of Scientific and Industrial Research-Institute of Microbial Technology (approval IAEC/16/12).

References

  1. Arya, S. B., Kumar, G., Kaur, H., Kaur, A. and Tuli, A. (2018). ARL11 regulates lipopolysaccharide-stimulated macrophage activation by promoting mitogen-activated protein kinase (MAPK) signaling. J Biol Chem 293(25): 9892-9909.
  2. Buscher, D., Hipskind, R. A., Krautwald, S., Reimann, T. and Baccarini, M. (1995). Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages. Mol Cell Biol 15(1): 466-475.
  3. Epelman, S., Lavine, K. J. and Randolph, G. J. (2014). Origin and functions of tissue macrophages. Immunity 41(1): 21-35.
  4. Gay, N. J., Symmons, M. F., Gangloff, M. and Bryant, C. E. (2014). Assembly and localization of Toll-like receptor signalling complexes. Nat Rev Immunol 14(8): 546-558.
  5. Platko, K., Lebeau, P. and Austin, R. C. (2018). MAPping the kinase landscape of macrophage activation. J Biol Chem 293(25): 9910-9911.
  6. Satoh, T. and Akira, S. (2016). Toll-like receptor signaling and its inducible proteins. Microbiol Spectr 4(6).
  7. Schaeffer, H. J. and Weber, M. J. (1999). Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 19(4): 2435-2444.
  8. Shapouri-Moghaddam, A., Mohammadian, S., Vazini, H., Taghadosi, M., Esmaeili, S. A., Mardani, F., Seifi, B., Mohammadi, A., Afshari, J. T. and Sahebkar, A. (2018). Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol 233(9): 6425-6440.
  9. Shimazu, R., Akashi, S., Ogata, H., Nagai, Y., Fukudome, K., Miyake, K. and Kimoto, M. (1999). MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med 189(11): 1777-1782.
  10. Sindhwani, A., Arya, S. B., Kaur, H., Jagga, D., Tuli, A. and Sharma, M. (2017). Salmonella exploits the host endolysosomal tethering factor HOPS complex to promote its intravacuolar replication. PLoS Pathog 13(10): e1006700.
  11. Zhang, X., Goncalves, R. and Mosser, D. M. (2008). The isolation and characterization of murine macrophages. Curr Protoc Immunol Chapter 14: Unit 14 11.

简介

巨噬细胞是高度吞噬细胞,其利用各种病原体识别受体(PRR)来识别病原体相关分子模式(PAMP)。这些PAMP可以存在于微生物中,例如细菌CpG DNA,并且被Toll样受体9(TLR9)识别,其是存在于巨噬细胞的内体膜上的PRR。 PAMP也可以存在于微生物的表面上,例如脂多糖(LPS),其装饰革兰氏阴性细菌的外膜,如鼠伤寒沙门氏菌和 Escherichia coli 。 LPS被存在于巨噬细胞质膜上的TLR4识别,并且LPS-TLR4结合导致信号级联的激活,包括MAPK磷酸化,其反过来促进巨噬细胞活化和微生物杀灭。该方案描述了用于研究目的基因在由原代骨髓衍生的巨噬细胞(BMDM)中的细菌感染诱导的细胞外信号调节蛋白激酶1和2(ERK1 / 2)信号传导中的作用的方法。

【背景】巨噬细胞是吞噬细胞,其可以作为库普弗细胞(在肝脏中)和腹膜巨噬细胞(在腹膜腔中)驻留到特定组织,或者可以响应于感染进入组织。巨噬细胞的主要功能包括吞噬和清除旧的受损细胞,以及血清中营养物质的循环(最近在Shapouri-Moghaddam评论 et al。,2018)。然而,对于巨噬细胞清除微生物,需要它们的活化。从许多组织如肺泡和腹膜腔获得的巨噬细胞包括大量预活化的巨噬细胞群。然而,源自骨髓中存在的骨髓祖细胞的巨噬细胞相对幼稚并且对激活刺激更敏感(Epelman 等人,2014)。在巨噬细胞遇到革兰氏阴性细菌时,存在于细菌表面或由血液中的细菌脱落的LPS被LBP(LPS结合蛋白)捕获并作为配体呈递给TLR4,I型跨膜蛋白存在于巨噬细胞的质膜上,其介导PAMPS的识别,例如LPS(Shimazu 等人,,1999)。 LPS与TLR4(以及其他共刺激分子)的结合导致在TLR4的细胞质尾部募集几种衔接蛋白,随后是细胞内事件的级联,导致核因子-κB(NF-κB)的活化和有丝分裂原激活蛋白激酶(MAPK)信号在TLR4下游级联,包括ERK1 / 2(Buscher et al。,1995; Schaeffer and Weber,1999; Gay et al。, 2014; Platko et al。,2018; Arya et al。,2018)。这些信号传导途径直接或间接地磷酸化和激活各种转录因子,这些转录因子导致参与促炎细胞因子,活性氧物质,亚硝化爆发和促进巨噬细胞活化的产生和释放的基因的表达(Satoh和Akira,2016)。因此,ERK1 / 2信号传导在增强巨噬细胞对细胞内病原体的反应中起重要作用。该方案描述了一种方法,用于研究宿主因子在调节原发性BMDM中ERK1 / 2激活对鼠伤寒沙门氏菌感染的作用中的作用(参见图1中的示意图和Arya et al。,2018)。

关键字:骨髓巨噬细胞, 沙门氏菌, 感染, ERK信号通路, 蛋白质印迹

材料和试剂

  1. 5毫升注射器(Dispovan)
  2. 60毫米组织培养皿(BD Falcon ®,目录号:353002)
  3. 10厘米组织培养皿(BD Falcon ®,目录号:353003)
  4. 6孔组织培养板(BD Falcon ®,目录号:353046) 
  5. 50毫升Falcon管(BD Falcon ®,目录号:352070)
  6. 15毫升Falcon管(BD Falcon ®,目录号:352096)
  7. 14毫升圆底聚丙烯管(康宁,目录号:352006)
  8. 1.5 ml微量离心管(MCT)(Tarson,目录号:50010)
  9. 0.2微米PVDF膜(Bio-Rad,目录号:162-077)
  10. X光胶片(Carestream,目录号:6574958)
  11. 铝箔
  12. 细胞刮刀(HiMedia,目录号:TCP104)
  13. 无菌22 G针(Dispovan)
  14. C57BL / 6雄性小鼠(通常6-8周龄)
  15. 鼠伤寒沙门氏菌菌株SL1344
  16. RPMI medium 1640(Lonza,目录号:12702F)
  17. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10082147)
  18. HEPES(1M)(Thermo Fisher Scientific,Gibco TM ,目录号:15630080)
  19. MEM非必需氨基酸溶液(100x)(Thermo Fisher Scientific,Gibco TM ,目录号:11140-050)
  20. 抗生素 - 抗真菌药(100x)(Thermo Fisher Scientific,Gibco TM ,目录号:15240-062)
  21. 丙酮酸钠(100 mM)(Thermo Fisher Scientific,Gibco TM ,目录号:11360-070)
  22. GlutaMAX TM 补充剂(Thermo Fisher Scientific,Gibco TM ,目录号:35050-061)
  23. DPBS(Thermo Fisher Scientific,Gibco TM ,目录号:14190-144) 
  24. PBS,pH 7.4(Thermo Fisher Scientific,Gibco TM ,目录号:10010-023) 
  25. 胰蛋白酶-EDTA(0.05%)(Thermo Fisher Scientific,Gibco TM ,目录号:25300-054) 
  26. 台盼蓝溶液(Thermo Fisher Scientific,Gibco TM ,目录号:15250-061) 
  27. 小鼠M-CSF(巨噬细胞集落刺激因子)(eBiosciences,目录号:34-8983)
  28. ACK(氯化铵 - 钾)裂解缓冲液(Thermo Fisher Scientific,Gibco TM ,目录号:A10492-01)
  29. 蛋白酶抑制剂(Sigma-Aldrich,目录号:P8340)
  30. 磷酸酶抑制剂(罗氏,目录号:4906837001)
  31. Bradford试剂(Sigma-Aldrich,目录号:B6916)
  32. TWEEN ® 20(Sigma-Aldrich,目录号:P9146) 
  33. Triton TM X-100(Sigma-Aldrich,目录号:T8787) 
  34. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771) 
  35. 脱氧胆酸钠(DOC)(Sigma-Aldrich,目录号:D6750)
  36. Trizma ®碱(Tris碱)(Sigma-Aldrich,目录号:T6066) 
  37. 氯化钠(NaCl)(西格玛奥德里奇,目录号:3014)
  38. 乙二醇 - 双(2-氨基乙基醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:3889)
  39. 脱水乙二胺四乙酸二钠盐(EDTA-Na 2 )(Sigma-Aldrich,目录号:E5134)
  40. 庆大霉素溶液(Sigma-Aldrich,目录号:G1272)
  41. 硫酸链霉素(Sigma-Aldrich,目录号:S6501)
  42. 乙醇(默克,目录号:108543)
  43. 脱脂牛奶(BD Difco,目录号:232100)
  44. LB肉汤(BD Difco,目录号:244620)
  45. SS琼脂(HiMedia,产品目录号:M108) 
  46. Laemmlisample buffer(Bio-Rad,目录号:161-0747)
  47. ON-TARGETplus非靶向对照siRNA池(Dharmacon,目录号:D-001810-10) 
  48. ON-TARGETplus Mouse Arl11 siRNA(Dharmacon,目录号:L-055672-01-0005)
  49. 多克隆抗总ERK1 / 2抗体(Cell Signaling Technology,目录编号:4695)
  50. 多克隆抗磷酸化ERK1 / 2抗体(Cell Signaling Technology,目录编号:4370)
  51. 过氧化物酶AffiniPure山羊抗兔IgG(H + L)(Jackson ImmunoResearch,目录号:111-035-144)
  52. ECL Plus Western Blotting Substrate(Pierce,目录号:32132)
  53. 70%乙醇(见食谱)
  54. 完整的RPMI培养基(参见食谱)
  55. 小鼠M-CSF原液(0.4 mg / ml)(见食谱)
  56. SS琼脂平板(见食谱)
  57. 链霉素原液(50 mg / ml)(见食谱)
  58. LB肉汤(见食谱)
  59. Opsonization缓冲区(参见食谱)
  60. 感染生长培养基(GM)(见食谱)
  61. RIPA裂解缓冲液(见食谱)
  62. 阻塞缓冲区(参见食谱)
  63. PBST(见食谱)
  64. 抗体稀释缓冲液(见食谱)

设备

  1. 烧杯
  2. 0.2微米聚醚砜滤料
  3. 剪刀和镊子(Fisher Scientific)
  4. 细胞培养CO 2 培养箱(Thermo Scientific,型号:371)
  5. 生物安全柜II级(ESCO,型号:AC2-4S8-NS)
  6. 倒置组织培养显微镜(尼康,型号:TS2) 
  7. 血细胞计数器(Sigma,目录号:Z359629)
  8. 多模阅读器(Tecan,型号:Infinite M200)
  9. 孵化器摇床(New Brunswick,型号:Innova 42) 
  10. 自动X光胶片处理器(Protec,型号:OPTIMAX 2010)
  11. 平板扫描仪(Microtek,型号:Bio-5000)
  12. 冷冻离心机带板式离心机桶,足以容纳6孔板(Eppendorf,型号:5810 R)
  13. 半干式水平吸墨系统(Atto,型号:WSE-4020)
  14. SDS-PAGE凝胶装置(Bio-Rad,型号:Mini Protean Tetra Cell)
  15. 跳舞振动筛(Tarson,型号:MC-02)
  16. 摇摆摇床(Tarson,型号:4080)
  17. 流式细胞仪

软件

  1. ImageJ软件(NIH, https://imagej.nih.gov/ij/ )
  2. Microsoft Excel软件(MS Office)

程序

该协议分为三个部分:A)小鼠原发性BMDM的产生; B)用鼠伤寒沙门氏菌感染BMDMs ; C)通过Western印迹评估感染沙门氏菌的巨噬细胞裂解物中活化/磷酸化水平ERK1 / 2。建议读者阅读图1所示的原理图,描述执行该协议的不同步骤。


图1.示意图,显示用于调查宿主因子在调节原发性骨髓衍生巨噬细胞(BMDM)中ERK1 / 2激活中的作用的步骤 鼠伤寒沙门氏菌 感染。分离小鼠骨髓细胞并在M-CSF存在下培养以产生原发性BMDM。在产生BMDM后,用针对目的基因的对照siRNA或siRNA转染细胞。 siRNA后处理,用鼠伤寒沙门氏菌的调理固定相培养感染BMDM。在感染后的不同时间结束时,进行细胞裂解并在SDS-PAGE凝胶上运行裂解物,进行Western印迹并使用化学发光底物在X射线胶片上显示信号。使用ImageJ软件量化扫描的X射线胶片的信号强度。


  1. 生成小鼠原发性BMDMs
    1. 准备三个60毫米组织培养皿,装满70%乙醇(见食谱1),DPBS或完整RPMI培养基(见食谱2),并在干净区域上放一对无菌(高压灭菌)镊子和剪刀,做好小鼠解剖准备在生物安全柜(BSC)内。另外,放置一块用70%乙醇清洗过的方形(12 x 12英寸)铝箔,作为鼠标解剖的平台。
      注意:在实验过程中始终保持无菌条件。
    2. 通过颈椎脱位牺牲小鼠并将小鼠浸入含有70%乙醇的烧杯中。使用小剪刀的尖端在鼠标的大腿附近切割,并使用剪刀和镊子从每个后肢的顶部向下剥离皮肤。通过切割尾骨和股骨(沿着与股骨感觉90°的骨头切割)将后肢从身体上分离。切断脚(t骨)和皮肤并丢弃。将去皮的肢体保持在含有70%乙醇的培养皿中5分钟,因为这会使肌肉脱水,使其更容易脱离骨骼(见图2A-2G)。
      注意:为了防止BMDM培养物被肌肉和成纤维细胞污染,去除肌肉。
    3. 使用剪刀去除四肢多余的肌肉,同时保持骨骼完整性。用剪刀轻轻按压以切断膝关节,切断股骨和胫骨之间的韧带(见图2H-2I)。将骨头置于70%乙醇中5分钟,然后将其转移至含有DPBS的培养皿中5分钟。
    4. 使用一对镊子将骨骼保持在中心,并使用剪刀切割胫骨/股骨的两端(距端部约3-6 mm)。用完全RPMI培养基填充5ml注射器,并通过骨的一端将骨髓(BM)冲洗到含有完全RPMI培养基的60-mm组织培养皿中。重复此步骤穿过骨的另一端以从骨的两端排出BM(图2J-2K)。
    5. 使用5ml注射器,将收集的BM通过22G针头3-4次以制备单细胞悬浮液,并将细胞收集在50ml Falcon管中(图2L-2N)。
    6. 在室温(RT)下将细胞悬浮液在700μM离心5分钟,并倾析上清液。
    7. 向细胞沉淀中加入1ml ACK裂解缓冲液以裂解RBC并继续轻微混合,孵育1分钟。立即加入10ml DPBS并在室温下以700×g离心离心细胞悬浮液5分钟(图2O)。
      注意:不要超过孵育时间超过1分钟,因为它可能会对RBC以外的细胞造成损害,并降低BMDM的总产量。
    8. 如步骤A6中所述,使用10ml DPBS洗涤细胞沉淀两次。
      注意:加入DPBS观察肌肉污染情况。如果存在任何肌肉碎片,请使用1 ml移液器将其取出。
    9. 最后一次洗涤后,将细胞沉淀重悬于15-20ml完全RPMI培养基中,并使用台盼蓝排除原理在倒置组织培养显微镜下用血细胞计数器计数细胞。
      注意:BM细胞的产量变化很大,因此应使用台盼蓝的不同稀释度来获得准确的细胞计数。通常,可以使用1:100台盼蓝的起始稀释液,孵育时间为1分钟进行细胞计数。
    10. 在10-cm组织培养皿中培养约4百万个BM细胞,该培养皿含有8ml补充有30ng / ml M-CSF的完全RMPI培养基(参见方法3)。将培养皿置于37℃和5%CO 2 的细胞培养箱中。将此步骤视为第1天。
    11. 观察第2天粘附的巨噬细胞百分比的增加。
    12. 在第3天,通过添加一半体积的最初使用的完整RPMI培养基补充BM培养物,即,4ml完全补充有M-CSF(30ng / ml)的RPMI培养基 
    13. 观察第4天粘附的巨噬细胞百分比(约60%)的增加。
    14. 到第5天,将观察到~90%粘附的巨噬细胞(图3)。要在功能实验中使用BMDM,请取出培养基,然后用10 ml DPBS轻轻洗涤细胞。要分离BMDMs,加入5 ml胰蛋白酶,将培养皿置于37°C培养箱中5-10分钟。孵育期后,快速查看显微镜下的细胞以确认脱离。接下来,通过添加5ml完全RPMI培养基进行胰蛋白酶中和。使用1 ml移液器吸头轻轻冲洗细胞,将细胞悬液转移到50 ml离心管中。
      注意:非常温和地执行步骤A14以避免激活巨噬细胞。
    15. 在室温下将细胞悬浮液在700μM离心5分钟离心,轻轻重悬于10ml完全RPMI培养基中,并使用血细胞计数器进行细胞计数,如步骤A9中所述。
    16. 在此步骤中,可以通过流式细胞术分析收集的细胞的巨噬细胞纯度,并准备进行进一步的表征和功能实验。
      注意:为了通过流式细胞仪分析分离的巨噬细胞的纯度,读者可以参考张 等。 ,2008。


      图2.用于制备BMDM的小鼠解剖。 A.通过颈椎脱位处死6-8周龄的C57BL / 6雄性小鼠,并在70%乙醇中灭菌并置于干净的铝上生物安全柜内的铝箔。是。使用小剪刀在鼠标大腿附近切割,并使用剪刀和镊子将每个后肢顶部的皮肤从脚上剥下来。 F.通过切割尾骨和股骨,将后肢从身体上分离。 G.切断脚(t骨)和皮肤。 H和I.使用剪刀从肢体上去除多余的肌肉,并通过用剪刀轻轻按压以切断股骨和胫骨之间的韧带来小心地分离膝关节。 J和K.用镊子将骨头固定在中心,用剪刀剪掉胫骨/股骨的两端。 L-N。使用5ml注射器,将收集的BM通过22G针头3-4次以制备单细胞悬浮液并在50ml Falcon管中收集细胞。 O.离心并将ACK裂解缓冲液加入细胞沉淀中以裂解RBC。立即DPBS并离心细胞悬液以收集BM细胞。


      图3.小鼠BMDM的表征。 在小鼠后肢分离的BM细胞在M-CSF存在下培养。在第5天,巨噬细胞群体很大程度上是粘附的。比例尺=100μm。

  2. 用鼠伤寒沙门氏菌感染BMDMs
    1. 在6孔组织培养板中,在每孔含有30ng / ml小鼠M-CSF的2ml完全RPMI培养基中,以1×10 6个细胞/孔的密度接种BMDM。所需的6孔组织培养板的数量将取决于要进行的siRNA处理的数量和待分析的不同感染时间点。 
    2. 第二天,用含有30ng / ml小鼠M-CSF的新鲜完整RPMI培养基替换培养基,并按照制造商的说明用对照siRNA和基因特异性siRNA转染BMDM。为了说明该方案,我们已经如Arya et al。,2018中所述进行了对照siRNA或Arl11 siRNA。
      注意:鼓励读者标准化条件 即 ,siRNA的数量和治疗时间,以达到他们感兴趣的基因的最大沉默。&nbsp ;
    3. 为了制备感染的静止培养物,从含有50μg/ ml链霉素的新鲜条纹SS琼脂平板(参见食谱4和5)中接种3ml LB肉汤中分离的鼠伤寒沙门氏菌 SL1344菌株的菌落(见配方6)在14ml圆底聚丙烯管中补充链霉素(50μg/ ml)。建立一夜之间的文化
      (~16小时)在37℃的振荡培养箱(200rpm)中。该步骤可在siRNA处理BMDM后56小时开始,以便在沙门氏菌培养物准备好感染时完成siRNA处理的持续时间(72小时)。
      注意: 鼠伤寒沙门氏菌 SL1344菌株对链霉素有抗性,在培养基中加入链霉素以防止任何其他污染物的生长。
    4. 测量O.D.使用分光光度计在600nm波长下过夜沙门氏菌培养物(在LB肉汤中稀释至1/10)。转移相当于O.D.的细菌培养物的体积。在MCT中= 1(~10 9 活细菌)。检查示例计算以估算O.D.所需的体积。 = 1.
      示例计算:
      如果O.D。 600 1/10稀释的过夜细菌培养物= 0.33
      因此,在1 ml培养物中计算的O.D. 600 细菌将= 0.33 x 10 = 3.3
      细菌数量等于10 9&nbsp; 1 ml, 即 ,OD 600&nbsp; = 1.0 < / em>
      因此,所需的细菌培养量=(1 /实际O.D. 600 )x 1 ml =(1 / 3.3)x 1 ml = 0.303 ml
      将0.303 ml细菌培养物转移到新鲜的MCT中,加入0.697 ml LB肉汤,使体积达到1 ml。
    5. 将稀释的沙门氏菌培养物在8,600 x g 离心3小时,在室温下离心,吸出0.9 ml LB肉汤并加入等体积的DPBS。通过温和涡旋重悬细菌细胞,再次重复离心步骤以除去LB肉汤。
      注意:离心1 ml后,仅吸出0.9 ml体积以防止细菌损失,并重复离心步骤两次,以完全去除LB肉汤。
    6. 进行细菌的调理作用。为了有效地粘附巨噬细胞表面上的细菌和吞噬作用,细菌表面涂有血清中存在的IgG。在步骤B5完成后吸出0.9ml DPBS,并将细菌沉淀重悬于900μl调理缓冲液中(参见方法7)。将细菌悬浮液在37℃孵育20分钟,间歇反转混合,然后在8,600 x g 离心3分钟。轻轻吸出上清液(约0.9 ml)并加入0.9 ml DPBS重悬细菌细胞沉淀。
      注意:离心1 ml后,仅吸出0.9 ml体积,以防止调理细菌的损失。
    7. 将调理后的细菌稀释至GM中的1/10(参见配方8),即,4.5ml GM(预热至37℃)+ 0.5ml调理细菌。这意味着,如果OD 600 = 1意味着1×10 9 细菌/ ml,那么1/10稀释将具有10 8 细菌/ ml。
      注意:步骤B6和B7应在单独的生物安全柜中进行,以尽量减少污染。
    8. 在感染复数(MOI)50:1下感染来自步骤B2的接种的BMDM(即,在72小时siRNA处理后)。要执行此操作,请删除完整的RPMI介质并使用DPBS清洗一次。然后快速将0.5ml来自步骤B7的稀释细菌加入每个孔中。 MOI是感染期间每个细胞添加的细菌数量。请参阅示例计算以估算实现MOI为50:1所需的体积。
      示例计算:
      BMDMs在6孔组织培养皿中以每孔10 6 细胞的密度接种 > 因此,对于MOI = 50:1,所需的细菌数量= 10 6 x 50 = 5 x 10 7
      我们在1毫升中有108个细菌(如步骤B7)
      因此,对于5 x 10 7 ,需要的培养体积= 1 ml x 5 x 10 7 / 10 8 =每孔0.5 ml细菌稀释液。
    9. 为避免BMDM对沙门氏菌的不均匀吞噬作用,通过在6孔组织培养板的每个孔中加入1.5 ml GM来同步该过程,用Parafilm密封板以避免溢出,并进行离心分离在30°C时453 xg
      5分钟。
    10. 取出Parafilm并在感染后(p.i.)样品将板转移至37℃和5%CO 2 的细胞培养箱中20分钟。

  3. 通过蛋白质印迹评估沙门氏菌感染的巨噬细胞裂解物中活化/磷酸化水平ERK1 / 2
    1. 从孔中吸出培养基以除去未内化的细菌,并用DPBS(1ml /孔)洗涤一次并完全除去DPBS而不移出细胞。&nbsp;
    2. 细胞裂解液的制备
      裂解未感染(对照)和20分钟p.i.样品加入冰冷的RIPA缓冲液,补充1x蛋白酶抑制剂和1x磷酸酶抑制剂(每孔0.1ml,6孔板)(参见方法9)。立即从平板上刮下细胞,将提取物转移到适当标记有样品名称的MCT中。保持在冰上。
    3. 向剩余的样品孔中加入2 ml GM(补充50μg/ ml庆大霉素),并将板转移至37°C和5%CO 2 的组织培养箱中进行下一次根据您的实验要求的感染间隔。例如,对于50分钟p.i.样品,将样品充分孵育30分钟。在每次p.i.结束时,如步骤C2中所述制备细胞裂解物。
      注意:在培养基中添加庆大霉素是为了防止即使在洗涤后遗留下来的非内化细菌的生长,庆大霉素对巨噬细胞的细胞毒性也很小。
    4. 将细胞裂解物保持在冰上最多10分钟(刮除后),然后在16,600 x g 4℃下离心10分钟。
    5. 在新鲜的MCT中小心地收集后核上清液(PNS)而不干扰沉淀。如果要在第二天进行SDS-PAGE,PNS可以储存在-80°C。
    6. 使用制造商的说明通过Bradford测定法定量PNS中存在的蛋白质浓度。将所有样品相对于彼此标准化以加载等量的蛋白质(每个泳道~15-20μg)。
    7. 标准化后,计算每个样品所需的体积并转移至新鲜MCT并加入Laemmli样品缓冲液至终浓度为1x,在99℃下煮沸7分钟,然后短暂旋转。
    8. 将样品加载到10%SDS-PAGE凝胶上并遵循标准Western印迹程序。
      注意:SDS-PAGE凝胶需要一式两份进行,因为针对p-ERK1 / 2和总ERK1 / 2的抗体检测到相同大小的条带。
    9. 在进行蛋白质转移步骤(30伏在冷室中2小时)后,将PVDF膜在封闭缓冲液(参见配方10)中在4℃下在水平振荡器上孵育过夜。
    10. 取出封闭缓冲液,用0.05%PBST洗涤膜三次,每次5分钟(见配方11)。&nbsp;
    11. 在抗体稀释缓冲液(0.05%PBST;参见配方12)中孵育膜和一抗(1:1,000稀释),在室温下温和搅拌2小时。
    12. 在舞动摇床上用0.05%PBST洗涤膜三次,每次5分钟。
    13. 将膜与山羊抗兔IgG(HRP-缀合的)抗体(1:5,000稀释)在0.05%PBST中孵育,在室温下温和搅拌1小时。
    14. 除去二抗溶液并在舞动摇床上用0.3%PBST洗涤膜三次,每次10分钟。
    15. 按照制造商的说明准备ECL试剂。用膜孵育ECL底物1分钟,去除多余的溶液(膜保持湿润),用塑料包裹并暴露于X射线胶片并在自动X光胶片处理器中显影信号(图4)。
    16. 使用平板扫描仪扫描显影的X射线胶片,并使用ImageJ软件对磷酸和总ERK1 / 2波段信号进行光密度定量,如下面的数据分析部分所述。


      图4.在感染 沙门氏菌 后,BMM中Arl11的消耗会损害ERK1 / 2活化。 Control-和Arl11-沉默的BMDM用鼠伤寒沙门氏菌感染不同的时间段,制备裂解物并用指定的抗磷酸-ERK1 / 2和抗-Ell-ERK1 / 2抗体印迹。

数据分析

使用ImageJ软件对磷酸和总ERK1 / 2信号进行光密度定量

  1. 1.启动ImageJ软件并打开磷酸化ERK1 / 2和总ERK1 / 2印迹的扫描图像,如图5A所示。转到“文件”→“打开”→“浏览”→“选择文件”→“打开”。
    2.从ImageJ工具栏中选择“矩形工具”,然后绘制一个覆盖通道1中带状信号的框,如图5B所示。转到“分析”→“凝胶”→“选择第一道”。
    3.将选择从第1道拖放到第2道,如图5C所示。然后转到“分析”→“凝胶”→“选择下一道”。重复此步骤,直到选中所有通道。
    4.要获得每个波段的像素强度,请转到“分析”→“凝胶”→“绘制通道”。然后从图像J工具栏中选择“魔杖工具”,将光标(+)置于第1道的直方图中心,然后对下一个窗格重复此步骤,并用鼠标左键单击以获得峰值下的区域(像素强度) )在一个单独的盒子中,如图5D所示。要执行此步骤,请确保选中“区域对话框”。要检查这一点,请转到“分析”→“设置测量”→“检查区域对话框”→点击“确定”。
    5.对于每个感染时间点,将测量值复制到MS Excel Sheet中,检测磷酸化ERK1 / 2和总ERK1 / 2印迹。然后计算每次p.i的磷酸-ERK1 / 2与总ERK1 / 2信号的比率。并绘制一个聚类列图。有关更多信息,建议读者参阅Arya et al。,2018年的图4a和4b。


    图5.使用ImageJ进行光密度定量。 显示了使用ImageJ软件进行光密度定量的不同步骤的屏幕截图。有关详细信息,请参阅协议的数据分析部分。

食谱

  1. 70%乙醇
    将70毫升无水乙醇与30毫升无菌蒸馏水(DW)混合
  2. 完整的RPMI媒体
    RPMI 1640
    10%热灭活FBS
    10 mM HEPES
    1 mM丙酮酸钠
    1x抗生素 - 抗真菌药
    1x MEM非必需氨基酸
    1x GlutaMAX TM 补充说明
    储存在4°C
  3. 小鼠M-CSF储备液(0.4 mg / ml)
    0.5 mg M-CSF
    1.25毫升PBS(pH 7.4)
    使用0.2微米聚醚砜过滤器过滤灭菌,等分并储存在-80°C
    注意:为了延长保质期(长达1年),在PBS(pH 7.4)中使用0.1%BSA作为溶剂。
  4. SS琼脂平板
    6.3克
    100毫升DW
    经常搅拌加热至沸腾以完全溶解介质。冷却至约45℃,加入所需浓度的抗生素,充分混合并倒入无菌培养皿中。将板保存在4°C
    注意:不要高压灭菌或过热SS-Agar培养基,因为它会破坏培养基的选择性。&nbsp;
  5. 链霉素原液(50 mg / ml)
    0.5克硫酸链霉素
    10毫升DW
    过滤灭菌,等分试样并储存在-80°C
  6. LB肉汤
    2.5克LB混合物
    100毫升DW
    高压灭菌并在室温下无菌条件下储存
  7. Opsonization缓冲区
    DPBS中的20%FBS
  8. GM感染
    RPMI 1640
    10%热灭活FBS
    10 mM HEPES
    1 mM丙酮酸钠
    1x MEM非必需氨基酸
    1x GlutaMAX TM 补充说明
    储存在4°C
    注意:在感染介质中不添加抗生素以防止内化细菌的停滞和杀灭。
  9. RIPA裂解缓冲液
    10mM Tris-Cl(pH 8)
    140 mM NaCl
    1 mM EDTA
    0.5 mM EGTA
    1%Triton TM X-100
    0.1%DOC
    0.1%SDS
    在RT商店
    注意:在立即使用前,将蛋白酶和磷酸酶抑制剂添加到RIPA裂解缓冲液中。
  10. 阻塞缓冲区
    含0.05%PBST的5%w / v脱脂奶粉
  11. PBST
    PBS中TWEEN ® 20所需的百分比
    通过搅拌充分混合并在室温下储存
  12. 抗体稀释缓冲液
    初级和二级抗体稀释(根据抗体数据表)在0.05%PBST中制成

致谢

该方案根据先前发表的研究进行了调整和修改(Sindhwani et al。,2017; Arya et al。,2018)。作者感谢理事会和科学理事会的财政支持。印度工业研究(CSIR)。 A. Tuli承认Wellcome Trust /生物技术部(DBT)的财政支持。这项工作得到了授予A. Tuli的Wellcome Trust / DBT印度联盟中级奖学金的支持(IA / I / 14/2 / 501543)。 A. Tuli还承认CSIR-微生物技术研究所(IMTECH,Chandigarh)的基础设施和财政支持(OLP-144,第038/2018号来文)。

利益争夺

作者声明没有竞争性的经济利益。

伦理

本研究严格按照动物实验监督委员会(1094 55/1999 / CPCSEA)根据1960年“防止虐待动物法”和1982年由动物部颁布的修正案发布的指导原则进行。环境与森林,印度政府。涉及小鼠实验的所有方案均由科学和工业研究委员会 - 微生物技术研究所的机构动物伦理委员会批准(IAEC / 16/12批准)。

参考

  1. Arya,S.B.,Kumar,G.,Kaur,H.,Kaur,A。和Tuli,A。(2018)。 ARL11通过促进丝裂素活化蛋白激酶(MAPK)信号传导来调节脂多糖刺激的巨噬细胞活化。 J Biol Chem 293(25):9892-9909。
  2. Buscher,D.,Hipskind,R.A.,Krautwald,S.,Reimann,T。和Baccarini,M。(1995)。 Ras依赖和非依赖性途径靶向巨噬细胞中丝裂原活化蛋白激酶网络。 Mol Cell Biol 15(1):466-475。
  3. Epelman,S.,Lavine,K。J.和Randolph,G.J。(2014)。 组织巨噬细胞的起源和功能。 Immunity 41( 1):21-35。
  4. Gay,N。J.,Symmons,M.F.,Gangloff,M。and Bryant,C.E。(2014)。 Toll样受体信号复合物的组装和定位。 Nat Rev Immunol 14(8):546-558。
  5. Platko,K.,Lebeau,P。和Austin,R。C.(2018)。 映射巨噬细胞激活的激酶情况。 J Biol Chem 293(25):9910-9911。
  6. Satoh,T。和Akira,S。(2016)。 Toll样受体信号传导及其诱导蛋白。 Microbiol Spectr 4(6)。
  7. Schaeffer,H。J.和Weber,M。J.(1999)。 丝裂原活化蛋白激酶:来自无处不在的信使的特定信息。 Mol Cell Biol 19(4):2435-2444。
  8. Shapouri-Moghaddam,A.,Mohammadian,S.,Vazini,H.,Taghadosi,M.,Esmaeili,SA,Mardani,F.,Seifi,B.,Mohammadi,A.,Afshari,JT and Sahebkar,A。( 2018)。 巨噬细胞可塑性,极化和健康与疾病的功能。 J细胞Physiol 233(9):6425-6440。
  9. Shimazu,R.,Akashi,S.,Ogata,H.,Nagai,Y.,Fukudome,K.,Miyake,K。和Kimoto,M。(1999)。 MD-2,一种赋予Toll样受体4脂多糖反应性的分子。 J Exp Med 189(11):1777-1782。
  10. Sindhwani,A.,Arya,S.B.,Kaur,H.,Jagga,D.,Tuli,A。和Sharma,M。(2017)。 沙门氏菌利用宿主内溶酶体系链因子HOPS复合物促进其腔内复制。 PLoS Pathog 13(10):e1006700。
  11. Zhang,X.,Goncalves,R。和Mosser,D。M.(2008)。 小鼠巨噬细胞的分离和鉴定。 Curr Protoc Immunol Chapter 14:第14单元11.
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Copyright: © 2018 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. Kumar, G., Arya, S. B. and Tuli, A. (2018). Method for Studying the Effect of Gene Silencing on Bacterial Infection-induced ERK1/2 Signaling in Bone-marrow Derived Macrophages. Bio-protocol 8(24): e3123. DOI: 10.21769/BioProtoc.3123.
  2. Arya, S. B., Kumar, G., Kaur, H., Kaur, A. and Tuli, A. (2018). ARL11 regulates lipopolysaccharide-stimulated macrophage activation by promoting mitogen-activated protein kinase (MAPK) signaling. J Biol Chem 293(25): 9892-9909.
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