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Jan 2020

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An in vitro DNA Sensor-based Assay to Measure Receptor-specific Adhesion Forces of Eukaryotic Cells and Pathogens
基于DNA传感器的真核细胞与病原体受体特异性粘附力的体外检测   

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Abstract

Motility of eukaryotic cells or pathogens within tissues is mediated by the turnover of specific interactions with other cells or with the extracellular matrix. Biophysical characterization of these ligand-receptor adhesions helps to unravel the molecular mechanisms driving migration. Traction force microscopy or optical tweezers are typically used to measure the cellular forces exerted by cells on a substrate. However, the spatial resolution of traction force microscopy is limited to ~2 µm and performing experiments with optical traps is very time-consuming.

Here we present the production of biomimetic surfaces that enable specific cell adhesion via synthetic ligands and at the same time monitor the transmitted forces by using molecular tension sensors. The ligands were coupled to double-stranded DNA probes with defined force thresholds for DNA unzipping. Receptor-mediated forces in the pN range are thereby semi-quantitatively converted into fluorescence signals, which can be detected by standard fluorescence microscopy at the resolution limit (~0.2 µm).

The modular design of the assay allows to vary the presented ligands and the mechanical strength of the DNA probes, which provides a number of possibilities to probe the adhesion of different eukaryotic cell types and pathogens and is exemplified here with osteosarcoma cells and Plasmodium berghei Sporozoites.

Keywords: Molecular force sensor (分子力传感器), DNA-hairpin (DNA发夹), Biomimetic surface (仿生表面), Receptor mediated forces (受体介导力), Molecular tension fluorescence microscopy (分子张力荧光显微镜), Plasmodium (疟原虫), Sporozoite (孢子体)

Background

Motile cells and pathogens interact in many different ways with their environment (Parsons et al., 2010; Nan, 2017; Muthinja et al., 2018). For example, transmembrane receptors anchor single cells in their environment and allow them to interact with other cells (Hynes, 1992). Integrins, which are the main class of receptors connecting cells to the extracellular matrix, transmit forces in a bidirectional manner (Schoen et al., 2013). On the one hand, extracellular mechanical signals are transduced to the cytosol, where they trigger signaling cascades and thereby control various cellular functions like proliferation, differentiation and migration (Wang et al., 1993; Janmey and Miller, 2011; Totaro et al., 2017). On the other hand, forces generated by the cytoskeleton can be applied to the extracellular environment, causing tissue shape changes and modulate cellular motility in specialized cells (Iskratsch et al., 2014).

To test for integrin-specific adhesion of cells, here, we first describe a method “option I” to prepare substrates presenting cyclic Arginin-Glycin-Aspartic acid (cRGD) ligands while non-specific surface interactions being prevented by a polyethylene glycol (PEG)-based passivation layer (Figure 1A). Of note, this method can be easily adopted to other receptors by varying the ligand.

To characterize the different aspects of cellular force transmission like absolute tension values, direction or the spatiotemporal distribution of forces several methods exist, which are based on the deformation of hydrogels, micropillars or unfolding of molecules, which differ in their spatial resolution and force range (Polacheck and Chen, 2016). One of the latest approaches visualizes traction forces exerted on single ligands via fluorescent DNA-based tension sensors (Wang and Ha, 2013; Blakely et al., 2014; Zhang et al., 2014). These double-stranded (ds) DNA or hairpin (particularly self-annealing single stranded (ss) DNA structures) sensors are immobilized on an in vitro surface and carry a ligand (e.g., cRGD) that enables for receptor (integrin)-specific cell interactions. Upon force, the dsDNA regions can be unfolded, which is detected with a fluorescent sensor (Figure 1B). In combination with standard wide-field or total internal reflection microscopy the spatiotemporal distribution of forces can be determined at high resolution, which is only limited by the optical resolution (~0.2 µm). The mechanical stability of dsDNA depends on its length and the GC content (Zhang et al., 2015). The force F1/2 that is required to unfold 50% of the DNA sensors, has been calibrated in previous single-molecule studies between ~4-60 pN for varying DNA sequences (Wang and Ha, 2013; Zhang et al., 2014).

The herein presented method “option II” builds on DNA hairpins, which allow to quantify single-molecular, force transmission in the range of 4-19 pN (Zhang et al., 2014; Liu et al., 2016). We modified the DNA strands to enable coupling of various ligands in a modular manner depending on the receptors of interest. In the closed configuration, the fluorescent tag is in close proximity to a quencher and thus dark. Above a specific threshold force the DNA-hairpins unzip and elongate, which lowers the quenching efficiency and gives rise to a digital ON/OFF signal (Figure 1B) (Woodside et al., 2006). They can refold if the force no longer persists (Bonnet et al., 1998; Woodside et al., 2006), which is accompanied by a decrease in fluorescence. This is especially important for the characterization of cells with high turnover of adhesion formations like motile pathogens. Here, DNA hairpin sensors were immobilized via gold nanoparticles, which serve as additional fluorescence quenchers. This reduces the background signals induced by thermodynamic fluctuations as well as the photobleaching in comparison to other sensor surfaces (Lang et al., 2004; Liu et al., 2016). If the range of forces a certain receptor can transmit is not previously known, it is advisable to start with the lowest force probe as it quantitively detects forces above its threshold. Next, the assay can be expanded by multiplexing sensors with different force thresholds and distinct fluorescent dyes to obtain semi-quantitative information on the distribution of cellular forces (Zhang et al., 2014).

Finally, we show how to combine substrate specific adhesion as established in “option I” with the molecular tension sensors “option II” allowing for traction measurements while assuring proper cell adhesion to the surface. This method “option III” can prevent frequent detaching of small and motile cells like Plasmodium sporozoites, even if they show low binding affinities for the ligands coupled to the force sensors.

By immobilizing the molecular tension sensors on such adaptable substrates, they can be used in a wide range of biophysical studies of cell adhesion to extracellular ligands. We recently employed molecular tension sensors to study the specific adhesion between pathogenic agents like viral particles and mammalian cells (Wiegand et al., 2020). Here we provide a protocol to study the interaction of different cell types, including Plasmodium sporozoites, with specific matrix ligands. The rapid movement of the Malaria parasites is a crucial part in the infection cycle (Frischknecht and Matuschewski, 2017) and has been investigated with traction force microscopy (Münter et al., 2009) and laser tweezers (Quadt et al., 2016). Note that the molecular sensors semi-quantitatively detect distribution of forces transduced via single receptors, while they do not report on the overall forces transmitted per cell (Goktas and Blank, 2017) nor the direction of force without integration of further fluorescence polarization techniques (Brockman et al., 2018). Since the methods are very sensitive to changes in the experimental procedure, we recommend to carefully control each step of the protocol.


Figure 1. Schematic of the experimental design and working principle of the in vitro DNA-sensor force assay. A. Step-by-step representation of the individual steps in this protocol to generate ligand-presenting surfaces (Opt. I), surfaces with DNA-based force sensors (Opt. II) and combination of cell adhesion ligands presented in the background of the surface and DNA-based force sensor bearing another type of ligand (Opt. III). Briefly, glass coverslips are cleaned, passivated with a PEG-layer and cellular ligands, gold nanoparticles and DNA strands are immobilized on top. For details, the indicated steps lead to the corresponding sections in the procedure chapter. B. Design of the DNA hairpin force sensors in relaxed conformation and under load. ‘Hairpin’ ssDNA (grey) forms the backbone of the sensors and is linked to the Gold nanoparticle at the bottom and a ligand at the top. Top ssDNA (orange) is linked to the fluorescent dye, bottom ssDNA (blue) is linked to a quencher. Additional bottom ssDNA (green) increases the quenching of background fluorescence. Annealing procedure of the DNA hairpin probe is described in Recipe 8. When a force is applied the partially self-annealing ‘hairpin’ strand unfolds by rupturing the hydrogen bonds between nucleotides, lowering the quenching efficiency between the two dyes, and thus local increase in the fluorescent signal. Figure is not drawn to scale.

Materials and Reagents

Standard

  1. 1.5 ml plastic reaction tube (e.g., Eppendorf, catalog number: 00 30125150 )
  2. Aluminum foil
  3. Cell hood
  4. Erlenmeyer flask of different sizes
  5. Flasks for buffers
  6. Glass beaker of different sizes
  7. Heat-resistant glass tub for oil bath
  8. Pipettes of different sizes with tips
  9. Schlenk flasks of different sizes
  10. Fine-tipped metallic tweezers, e.g., #4, #7 (Sigma, catalog number: T6165 )
  11. Ice
  12. Ultra-pure deionized water max. resistance of 18.2 MΩ·cm, e.g., MilliQ purified
  13. Inert gas (Ar)
  14. Nitrogen gas
  15. Schlenk line (Schlenk flask with wide opening, lid for Schlenk flask, connection tubing for Vaccuum and Nitrogen gas)
  16. Deionised (DI) water

Consumables

  1. 20 x 20 mm coverslips #1 (Carl Roth, catalog number: H873.2 )
  2. DWK Life Sciences DURANTM Filter Funnels (No. 3) (Fisher Scientific, catalog number: 09-841-090 )
  3. GE Healthcare illustraTM, NAP-5 column (Fisher Scientific, catalog number: 10054394 )
  4. Glass coverslip holder (home-built)
  5. Kimwipe tissues (Kimberly-Clark Worldwide, catalog number: 0 5517 )
  6. Molecular sieve 3 Å (Carl Roth, catalog number: 8487 )
  7. Pasteur pipettes ISO 7712 (230 mm) (Karl Hecht, catalog number: 40567002 )
  8. Petri dish, PS, 35/10 mm with vents (Greiner Bio-One, catalog number: 627102 )
  9. Rotilabo-syringe filter, CME, sterile (Carl Roth, catalog number: P818.1 )
  10. Silicon chamber (Ibidi GmbH, catalog number: 80841 )
  11. Teflon coverslip holder (home-built, commercially available, e.g., at Thermo Fisher Scientific, catalog number: C14784
  12. Silicon glue‚ Twinsil 22, Addition-curing duplucating silicone (Picodent GmbH, catalog number: 13001000 )
  13. Silicon oil for oil bath (Sigma-Aldrich, catalog number: 85409 )

Chemicals

  1. Gold Nanospheres, 9 nm (nanoComposix, custom size, 9 ± 2 nm, 0.05 mg ml−1 in tannic acid)
  2. (+)-Sodium L-ascorbate (Sigma-Aldrich, catalog number: A7631 )
  3. 3-(triethoxysilyl)propylisocyanate (Sigma-Aldrich, catalog number: 413364 )
  4. Acetic acid ethyl ester (Carl Roth, catalog number: 6784 )
  5. Acetonitrile (CHROMASOLV for HPLC) (Honeywell International, catalog number: 60-002-31 )
  6. Aminoguanidine hydrochloride (Sigma-Aldrich, catalog number: A8835 )
  7. ɑ-Silane-ω-alkyne PEG, (CH3O)3-Si-PEG(3000)-C≡CH (Rapp Polymere, catalog number: 133000-70-71 )
  8. ɑ-Silane-ω-azido PEG, (CH3O)3-Si-PEG(3000)-N3 (Rapp Polymere, catalog number: 133000-5-71 )
  9. Copper sulfate (Sigma-Aldrich, catalog number: C1297 )
  10. Diethyl ether (Sigma-Aldrich, catalog number: 309966 )
  11. Dimethyl sulfoxide (for spectroscopy) (DMSO) (Merck, catalog number: 102950 )
  12. cRGD-alkyne (Biotrend Chemicals, custom synthesis: Cyclo(Arg-Gly-Asp-D-Phe-Pra))
  13. Hydrogen peroxide 30% (Merck, catalog number: 107209 )
  14. LA-PEG(3400)-NH2 (Biochempeg Scientific, catalog number: HE039005-3.4K )
  15. Methanol (Carl Roth, catalog number: 00 82 )
  16. N,N-Dimethylformamide (anhydrous, 99.8%) (DMF) (Sigma-Aldrich, catalog number: 227056 )
  17. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  18. Potassium hydroxide (for cleaning bath) (Sigma-Aldrich, catalog number: P5958 )
  19. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  20. QSY21 (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q20131 )
  21. Silane-PEG-methoxy, Sil-PEG(2000)-m (Biochempeg Scientific, catalog number: MF001020-2K )
  22. Sodium carbonate (Na2CO3) (Carl Roth, catalog number: A135 )
  23. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  24. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: 255793 )
  25. StarRed-NHS (Abberior) (Abberior, catalog number: 1-0101-011-3 )
  26. Sulfuric acid 96% (VWR CHEMICALS, catalog number: 1.08131.1000
  27. Toluene (Merck, catalog number: 108325 )
  28. Triethylamine (Sigma-Aldrich, catalog number: 471283 )
  29. Triethylammonium acetate (TEAA) (Sigma-Aldrich, catalog number: 69372 )
  30. Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) (Sigma-Aldrich, catalog number: 762342 )
  31. Click reaction mixture (CuAAC) (see Recipes)
  32. Dried toluene (see Recipes)
  33. Sil-PEG(3500)-LA (see Recipes)

DNA strands (custom synthesis by Integrated DNA Technologies, Inc.)
Note: Self-annealing sections underlined, top strand annealing dotted, bottom strand annealing dashed; a list of the hairpins used in literature and their calibration can be found in Zhang et al., 2014 and Liu et al., 2016.

  1. Top: 5′- /5AmMC6/ CGC ATC TGT GCG GTA TTT CAC -3′ 
  2. Bottom NH2-quencher:
    5′- /5ThioMC6-D/TTT GCT GGG CTA CGT GGC GCT CTT/3AmMO/ -3′
  3. Hairpin 4.2 pN:
    5'- /5AzideN/GTG AAA TAC CGC ACA GAT GCG TTT - GTA TAA ATG TTT TTT TCA TTT ATA C - TTT AAG AGC GCC ACG TAG CCC AGC AAA/3ThioMC3-D/ -3’ 
  4. Hairpin 12 pN:
    5'- /5AzideN/GTG AAA TAC CGC ACA GAT GCG TTT - GGG TTA ACA TCT AGA TTC TAT TTT TAG AAT CTA GAT GTT AAC CC - TTT AAG AGC GCC ACG TAG CCC AGC AAA/3ThioMC3-D/ -3′ 
  5. Hairpin 19 pN:
    5'- /5AzideN/GTG AAA TAC CGC ACA GAT GCG TTT - CGC CGC GGG CCG GCG CGC GGT TTT CCG CGC GCC GGC CCG CGG CG - TTT AAG AGC GCC ACG TAG CCC AGC AAA/3ThioMC3-D/ -3′
  6. Labeled DNA strands (see Recipes)
  7. Annealed DNA strands (see Recipes)

Enzymes

  1. StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific, Gibco®, catalog number: A1110501 )

Medium

  1. Albumin Fraction V (Carl Roth, catalog number: 8076 )
  2. Fetal Bovine Serum (USA) (Sigma-Aldrich, catalog number: F2442 )
  3. MEM α (Thermo Fisher Scientific, Gibco®, catalog number: 12561 )
  4. Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Gibco®, catalog number: 15140148 )
  5. RPMI 1640 Medium (Thermo Fisher Scientific, Gibco®, catalog number: 11-875 )
  6. Sodium Pyruvate 100 mM (100x) (Thermo Fisher Scientific, Gibco®, catalog number: 11360070 )
  7. Microscopy U2OS medium (see Recipes)
  8. Microscopy sporozoite gliding medium (see Recipes)
  9. PBS buffer (see Recipes)

Cell lines

  1. Plasmodium berghei ANKA strain (BEI resources (ATCC), catalog number: MRA-871 )
  2. Plasmodium berghei NK65 strain, CS-GFP line (optional usage, see Step H4) (not commercially available, requestable from cooperating parasitological institutes; further information can be found in Natarajan et al., 2001)
  3. U2OS, osteosarcoma cells (ATCC, catalog number: HTB-96 )

Equipment

  1. 300 °C Oven
  2. Freezer (-20 °C and -80 °C)
  3. Fridge (4 °C)
  4. Fume hood
  5. Centrifuge (Thermo Fisher Scientific, model: Heraeus Biofuge Pico )
  6. Centrifuge (Thermo Fisher Scientific, model: Heraeus Multifuge 1S-R ; catalog number: 75004331 )
  7. Hot Plates Accessories (IKA, model: ETS-D5 , catalog number: 0003378000 )
  8. HPLC (GE Comp Healthcare Life Sciences, model: ÄKTA Pure Chromatography System )
  9. HPLC column (Phenomenex, model: Luna 5u C18(2)-RP-HPLC , catalog number: 00G-4252-N0 )
  10. Lyophilisator (Labconco, model: FreeZone Plus 2.5 Liter Cascade Benchtop Freeze Dry System , catalog number: 7670020 )
  11. Magnetic Stirrers (IKA, model: C-MAG HS 7 , catalog number: 0003581200 )
  12. Wide-field, inverted fluorescence microscope (Applied Precision, model: DeltaVisionTM Elite Microscope System, based on an Olympus IX71 body) with home-built environmental chamber (commercially available incubators e.g., at OKOLAB)
  13. Microscope filter set (Applied Precision, model: DeltaVisionTM Elite Filter Sets)
  14. Microscope objective (Olympus, model: Objective 60x/1.4 NA PlanApo IX70 )
  15. Sonicator (EMAG Technologies, model: Emmi-H22 , catalog number: 9 82 17-DE)
  16. Spectrophotometer (NanoDrop Technologies, model: PEQLAB Nano Drop ND-1000 )
  17. Thermal cycler (Bio-Rad Laboratories, model: Bio-Rad T100 , catalog number: 1861096 )

Software

  1. Fiji is just ImageJ (Version 1.52e; https://fiji.sc/) (Schindelin et al., 2012)
  2. Stack contrast adjustment, plugin for Fiji (Michálek et al., 2007)

Procedure



Figure 2. Overview of the experimental procedure. Glass coverslips are handled with a pair of tweezers and inserted in a Teflon holder (white), which is transferred into a beaker with Piranha solution for cleaning (Procedure A). For passivation of the coverslips they are transferred into a glass holder and incubated with the Sil-PEG passivation solution in a Schlenk flask at 80 °C in an oil bath. The Schlenk flask can be attached to a vacuum and nitrogen line via its valve to provide an inert reaction container (not shown, Procedure B). After cleaning and drying the coverslips, specific ligands and gold nanoparticles (red solution) are immobilized by incubating a drop of the respective solution in-between two coverslips. To remove the coverslips again from each other, additional water or PBS is injected with a pipette and the upper coverslip is lifted with a pair of tweezers (Procedures C-E). To observe the surfaces at an inverted microscope they are glued with a two-component silicon glue (yellow and blue gel) into a Petri dish with a custom-made hole in the center (2 mm smaller than the coverslip) (Procedure F). The final device is shown in the last figure, where medium and cells can be added before transferring to a microscope. Further information is provided at the steps indicated below the pictures.


  1. Cleaning of glass surfaces
    1. Clean and activate coverslips in a Teflon holder in ~100 ml freshly prepared ‘Piranha solution’ [3:1 (v/v) sulfuric acid: hydrogen peroxide] for 1 h. Make sure the coverslips are completely covered by solution (Figure 2, cleaning). Stir the Piranha solution e.g., with an acid-resistant Pasteur pipette.
      CAUTION: Piranha solution is very corrosive and reacts exothermic. It must not get in contact with organic solvents. Perform every work step containing Piranha solution under a fume hood with constant ventilation.
      Note: From here on transfer and handle the coverslips only with tweezers (Figure 2).
    2. Remove ‘Piranha solution’ from the coverslips by carefully decanting it into an appropriate waste container according to the safety regulations of your institute, e.g., discarding after 10-20x diluted in water.
    3. Rinse coverslips 3 times with DI water and twice with ultra-pure water.
    4. Sonicate coverslips in ultra-pure water once for 5 min in ultrasonic cleaner at ~60 watt (standard sonication power).
    5. Dry surfaces in a nitrogen stream by holding them carefully with tweezers.
      Check point: Observe by eye that no stripes or drops of water remain else repeat Steps A3-A4 or discard the coverslip.

  2. Passivation of glass surfaces
    1. Transfer dried coverslips to a glass holder with tweezers and avoid touching them in the center.
    2. Prepare passivation solution as indicated in Table 1 (depending on the desired surface Opt. I-III, see Figure 1A) under continuous protective nitrogen atmosphere in a Schlenk flask (Figure 2, passivation). All steps involving organic solvents (Steps B2-B7) shall be performed following the safety regulations of your institute under a fume hood with constant ventilation. Create a protective atmosphere by connecting the Schlenk flask to a vacuum line and nitrogen supply. First vacuum-empty the flask, then flush with nitrogen and reduce the nitrogen stream in the following to add the powdery PEG, dried toluene and triethylamine.

      Table 1. Passivation compositions for the three different type of surfaces (Option I-III). Option I should be chosen when the adhesion of cells to a specific ligand is to be tested, Option II to access the forces at such specific ligands and Option III when ligand-specific adhesion to the surface should be granted independent of the force measurements of specific ligands.


    3. Add coverslips under protective atmosphere into the Schlenk flask in the solution and close the lid and gas supply. The reaction is now kept under protective atmosphere.
    4. Heat tube overnight at 78-80 °C in an oil bath. The oil bath can be set up combining the heat-resistant glass tub filled with oil on the magnetic stirrer. The additional thermostat keeps the temperature constant.
    5. Wash passivated coverslips by sonication in ethyl acetate for 5 min within their glass holder.
    6. Wash passivated coverslips by sonication in methanol for another 5 min within their glass holder.
      Pause point: It is possible to store passivated glass surfaces in methanol for up to 5 days if they are not used immediately.
    7. Dry surfaces in a nitrogen stream by holding them carefully with tweezers. Nitrogen stream can be dosed carefully with a compressed air gun. Make sure not to scratch the passivated surfaces during the entire procedure.
      Check point: As control for proper passivation add a dye (e.g., 100 nM StarRed in PBS) or seed adhesive cells on a passivated versus a non-passivated coverslip and observe their attachment to the surface under a microscope.
    8. For reuse, clean the used Schlenk flask and glass sample holder from PEG residue in a potassium hydroxide bath overnight and wash them afterwards with DI water.

  3. Option I–Immobilization of specific cellular ligands
    1. Prepare 100 µl of the reaction mixture for copper-catalyzed azide-alkyne cycloaddition (CuAAC, see Recipe 7) containing the desired cellular ligand.
      Note: Choose appropriate alkyne or azide functionalized PEG for azide or alkyne-ligands, respectively.
    2. Prepare a humidity chamber with parafilm on the bottom and a wet tissue inside a Petri dish where coverslips are placed onto (Figure 2, immobilization). Parafilm is used to hold the coverslips in their spot and to ease the separation of ‘sandwiched’ glasses later on.
    3. Place a drop of 100 µl click mixture on top of a passivated and dried coverslip. 
    4. Place a second, identical coverslip on top of the first one.
    5. Incubate the ‘sandwich’ for 2 h at room temperature (RT).

  4. Option II–Immobilization of DNA hairpin force sensors
    1. Place a drop of 250-300 µl of the gold nanoparticle solution on top of a passivated and dried coverslip.
    2. Incubate the coverslips for 30 min at RT.
    3. In the meantime, hybridize hairpin force sensors using ‘top’ and ‘bottom’ and ‘hairpin’ ssDNA. Use DNA mixture and annealing program as indicated (see Recipe 8). If DNA strands are purchased unlabeled, DNA and fluorophore should be coupled in a previous step (see Recipe 4 for labeling of DNA strands below).
    4. Separate the surfaces by carefully pipetting 300 µl ultra-pure water in-between the glasses and pick them up with a pair of tweezers (Figure 2, Immobilization). Clean them from supernatant gold nanoparticle solution by thoroughly rinsing with ~10 ml ultra-pure water.
      Check point: Confirm successful immobilization of gold nanoparticles by the pink shine of the coverslip.
    5. Remove excess liquid with tissues over the coverslip edges without letting them run completely dry or touching them.
    6. Place a drop of 100 µl DNA mixture on top of a gold-coated coverslip.
    7. Place a second, identical coverslip with the gold nanoparticle-coated side facing down on top of the first one.
    8. Incubate the ‘sandwich’ overnight at 4 °C under exclusion of light.
    9. Prepare 100 µl of the reaction mixture for CuAAC (see Recipe 7) containing tripeptide/ligand.
    10. Separate and clean the surfaces carefully from DNA mixture by washing in PBS (analogous to Step D4).
    11. Remove excess liquid with tissues over the coverslip edges without letting them run completely dry or touching them.
    12. Place a drop of 100 µl click mixture on top of a DNA-coated coverslip.
    13. Place a second, identical coverslip with the DNA-coated side facing down on top of the first one.
    14. Incubate the ‘sandwich’ for 2 h at RT in the humidity chamber under exclusion of light to avoid bleaching of the fluorophores.

  5. Option III–Combination of specific cellular ligand and DNA hairpin force sensors
    1. Prepare 100 µl of the 1st reaction mixture for CuAAC (see Recipe 7) containing 1st ligand.
    2. Prepare a humidity chamber with a wet tissue inside a Petri dish and flat parafilm on the bottom where coverslips are placed onto.
    3. Place a drop of 100 µl click mixture on top of a passivated and dried coverslip.
    4. Place a second, identical coverslip on top of the first one.
    5. Incubate the ‘sandwich’ for 2 h at RT.
    6. Separate and clean the surfaces carefully from click mixture by washing in PBS (analogous to Step D4).
    7. Further steps are equal to ‘D) Immobilization of DNA hairpin force sensor immobilization’ Steps D1-D14 using the 2nd ligand at Step D9.

  6. Sample preparation for microscopy
    1. Separate and clean the surfaces carefully from click mixture by washing in PBS (analogous to Step D4).
    2. Remove excess liquid with tissues over the coverslip edges without letting them run completely dry or touching them.
    3. Glue coverslips with the functionalized surface facing up into Petri dishes with custom made holes (2 mm smaller than the coverslips) in the bottom. Therefore, mix ~200 µl of Twinsil 22 silicon 1:1 e.g., with a pipette tip and apply the glue on the edges of the hole, such as the coverslip completely covers the hole and is surrounded by glue.
    4. Wait curing time of the glue (~ 5 min, check with the remaining glue ). If necessary, clean surface by washing in PBS to remove glue contamination.
      Check for successful immobilization of cellular ligands and force sensors by comparing cellular attachment (Procedure G) and homogenous background fluorescence, respectively, of only passivated coverslips versus those with immobilized ligands (Procedure C) or force sensors (Procedure D).

  7. Assays with cells (U2OS)
    1. Bring chamber for cell observation to 37 °C and 5% CO2 atmosphere.
    2. Fill 2-3 ml cellular medium (0.5% FBS) (see Recipe 5) in the Petri dishes prepared in step F covering the functionalized coverslip. Use medium with low FBS content to prevent unspecific ligand interactions and improve microscopy. Use a cell culture fume hood to prevent medium or cell culture contamination.
    3. Harvest adherent cells from culture flask by washing with PBS and detach with Accutase following standard protocols (e.g., Basic Cell Culture Protocols (Helgason and Miller, 2004)).
      Note: Accutase gently detaches cells for faster spreading results.
    4. Seed cells in diluted concentration to allow an undisturbed spread. In case of U2OS 105 cells reach an adequate density (calculated from T-25 and 35 mm dish flask surface for around 1:10 dilution).
    5. Wait until cells settle down and start productively spreading on the surfaces (10-30 min).
    Note: Force transmission is observed best 30 min post seeding cells and appears as bright dots and radial stripes of opened DNA hairpin sensors (Figure 3).


    Figure 3. U2OS cells adhering and exerting forces on specific ligand presenting surfaces. Representative bright-field, interference reflection microscopy (IRM) and fluorescence wide-field images of U2OS cells 15-20 min after seeding on the surfaces with (A) option I: cRGD-functionalization and (B) option II: cRGD-linked DNA hairpin sensors (4.2 pN opening force; StarRed-fluorophore). Scale bars = 10 µm.

  8. Assays with sporozoites
    1. Try to keep the edges of the coverslip as dry as possible, whereas the center of the functionalized surface should be kept humid. This eases the silicon chamber founding.
    2. Press a silicon chamber with its sticky side on the edges of the functionalized coverslip to minimize the cellular distribution area (Note: silicon sticks best on dry glass). Purchased multi-well chambers can be cut to a fitting size. This step can be skipped, but eases to generate an adequate parasite density.
    3. Fill 15 µl sporozoite medium (6% BSA) (see Recipe 6) into the silicon chamber.
    4. Add 15 µl ice-cooled PBS containing freshly isolated sporozoites from 6-8 pairs of well infected mosquitoes’ salivary glands (for a detailed protocol of mosquito infection and sporozoite collection see Prinz et al., 2017) resulting in 3%-BSA-medium.
      Notes:
      1. FBS-reduced cellular medium (0.5% FBS) can be use as well.
      2. Do not keep salivary glands on ice for prolonged times as this could harm the sporozoite motility.
      3. Usage of GFP-tagged sporozoites eases the selection of well infected salivary glands as well as their isolation. They can be employed on ligand-presenting surfaces (option I, see Figure 4A), but care must be taken to choose proper filter sets when using them together with force sensors (Figure 4B) to avoid fluorescent bleach through.
    5. Spin cells down to surface in a centrifuge at 2,000 x g for 3 min. Well-founded silicon chamber can stand this procedure. 
    6. Motility can be observed directly after attaching to the surfaces for around 30 min (Figure 4).


      Figure 4. Sporozoites (P. berghei) gliding on specific ligand presenting surfaces. Representative bright-field, interference reflection microscopy (IRM) and fluorescence wide-field time lapse images of (A) GFP-expressing sporozoites 5 min after seeding on the surfaces with option I: cRGD-functionalization and (B) WT sporozoites 5 min after seeding on the surfaces with option II: cRGD-linked DNA hairpin sensors (4.2 pN opening force; StarRed-fluorophore). For Option II Osteosarcoma cells (hollow arrow) were seeded together with the sporozoites as a positive control for the DNA-based force sensors (see Figure 3). Sporozoites adhere and glide on both surfaces, however, no tension signal was detected with the DNA hairpin sensors. This might be due to sub-threshold forces, few surface interactions or too short adhesion times (sporozoites move at ~2 µm/s), which do not allow for detectable unfolding of the tension probes, while osteosarcoma cells adhering on the same substrates gave rise to fluorescent signals (hollow arrow). Bright fluorescent spots (full arrow) are auto fluorescent remnants from the purification of Sporozoites out of mosquitoes’ salivary glands. Scale bars = 10 µm.

  9. Microscopy settings
    1. Imaging was performed on a fluorescence wide-field microscope (Olympus) equipped with a custom-made environmental chamber for cellular conditions (37 °C; 5% CO2). Sporozoites were usually imaged at RT without CO2 supply but we also successfully performed gliding motility assays under cellular conditions.
    2. Brightfield images were taken with a polarized light filter set.
    3. Fluorescence microscopy images were taken with excitation bandpass filter 632/22 nm and emission filter 679/34 nm.
    4. Interference reflection microscopy (IRM) images were taken with excitation bandpass filter 542/27 nm in IRM mode (set emission bandpass filter 525/48 nm) with minimal exposure time and intensity. IRM observation can be thus used as a low-bleaching control for cell adherence to the surface during the imaging process.

Data analysis

Receptor surfaces (option I), which do not contain force sensors, can be used for a qualitative testing of the cellular adherence properties to defined ligands on an otherwise non-adherent, PEG-ylated surface. Brightfield imaging or IRM allows to validate if cells adhere tightly to the surface, such as no fluorescence microscopy is required for this approach.
    Furthermore, quantitative analysis of the settling and spreading time of cells on the surface (data analysis see Schaufler et al., 2016) or of the gliding motility in case of motile sporozoites (data analysis see Prinz et al., 2017) can be used for comparable data analysis between different cell/parasite lines or different ligands.
    DNA force sensor surfaces (options II and III) provide a digital ON-OFF signal at a specific threshold force. Fluorescence microscopy images of the sensor surface provide spatial, temporal, and semi-quantitative information on the exerted forces, indicated by the transient appearance (hairpins) of the fluorescent signal. Therefore, fluorescence microscopy images were first adjusted for brightness and contrast for better visibility of the signals with the Fiji software. For time lapse imaging, ‘Stack contrast adjustment’ plugin is used to correct for the photobleaching. A semi-quantitative force readout can be generated from the local brightness reflecting the relative amount of dsDNA opening and displayed via a color-coded LUT e.g., “spectrum” (for more details see Zhang et al., 2014, Supplementary Figure 7). However, this quantitative analysis is limited by the optical resolution, since typically multiple sensors will be contained per diffraction limited spot.
    To narrow down the absolute traction values, the experiment can be repeated with DNA sensors of different rigidity by varying the DNA sequence. Multiple of these sensors coupled to different fluorophores can be combined on one surface for multiplex results (see Zhang et al., 2014).

Notes

  1. Prepare aliquots of appropriate volumes for frozen medium or solution to avoid repeated freeze thaw cycles and elongate their shelf lifetime.
  2. Separate and clean the surfaces from reaction mixture by carefully adding 300 µl PBS inside the sandwich with a pipette. Subsequently lift off top coverslip and wash them in PBS. Make sure you never scratch the surfaces during the entire procedure.
  3. Avoid drying out of the surfaces at any time since retracting liquids can produce artefacts caused by surface tension on surfaces.
  4. Other fluorescent dyes and quenchers with NHS function can be used for the labeling of DNA depending on the availability of filters at the microscope. Consider dyes with a high quantum yield and an emission wavelength close to the particle resonance peak of gold nanoparticles for stronger quenching (Kang et al., 2011). Note, that cellular autofluorescence, especially from nucleus, can overlap sensor signals from underneath the cell. For smaller cells with a high nuclear to body ratio (e.g., Plasmodium sporozoites), far-red dyes like StarRed can minimize this signal-autofluorescence interference.

Recipes

  1. PBS buffer
    1. Prepare PBS as indicated:
      NaCl            8.006 g (cfinal: 137 mM)
      KH2PO4      1.375 g (cfinal: 10.1 mM)
      KCl              201.3 mg (cfinal: 2.7 mM)
      Na2HPO4    255.5 mg (cfinal: 1.8 mM)
    2. Add 0.8 L ultra-pure water
    3. Adjust pH to pH 7.4 by adding a few Milliliter HCl (1 M)
    4. Add ultra-pure water to a total volume of 1 L
    5. Store buffer at RT
  2. Sil-PEG(3500)-LA
    1. Solve 145 µmol NH2-PEG(3400)-LA in 3 ml DMF in a small Schlenk flask under nitrogen atmosphere with a magnetic stir bar
    2. Add 159.5 µmol 3-(trimethoxysilyl)propylisocyanate (1.1-times the molecular amount of PEG)
    3. Stir overnight at room temperature
    4. Cool solution down to 4 °C
    5. Add cooled diethyl ether for 1 h
    6. Wash the reactant with cooled diethyl ether over a glass frit (No. 3)
    7. Dry gained Sil-PEG-LA from frit overnight under vacuum
    8. Store the powder at -20 °C under inert gas
  3. Dried toluene
    1. Dry molecular sieve thoroughly in an oven
    2. Vacuum an empty Erlenmeyer tube and add a nitrogen atmosphere
    3. Add toluene under protection atmosphere and close flask properly
    4. Wait several hours to get rid of excessive water in the toluene
    5. Store at room temperature under nitrogen atmosphere
  4. Labeling of DNA strands
    1. Dissolve DNA in ultra-pure water to a concentration of 1 mM
    2. Dissolve dye-NHS respectively quencher-NHS in dried DMSO to 10 mM
    3. Dissolve 105.99 mg Na2CO3 in 1 ml ultra-pure water
    4. Mix 60 µl solved DNA top strand (cfinal: 0.2 mM), 60 µl solved dye-NHS (cfinal: 2 mM) and 30 µl Na2CO3 solution (cfinal: 0.1 mM) in 150 µl PBS. Proceed the same way with DNA bottom strand and the quencher-NHS
    5. Adjust pH to 8.5
    6. Stir mixture overnight at room temperature under the exclusion of light
    7. Purify mixture in NAP-5 column as indicated in the user manual of the manufacturer
    8. Purify mixture in HPLC (gradient high-pressure liquid chromatography). Use Luna 5u C18(2)-RP-HPLC column, mobile phases are trietyhlammonium acetate (TEAA) (0.1 M) and acetonitrile with a gradient from 10% to 50% in 90 min and from 50% to 90% in 20 min
    9. Collect fraction showing both maximum absorption peaks at the same time for DNA (260 nm) and dye
    10. Freeze product to -80 °C and lyophilize it after that to get rid of the solvents
    11. Resuspend pure product in ultra-pure water
    12. Determine the concentration with a nanodrop spectrophotometer measuring the absorbance ADNA; 1mm of DNA at 260 nm and the absorbance Adye; 1mm at the excitation wavelength of the respective dye/quencher
    13. Calculate concentration from three different measurements via:


      Note: Take the extinction coefficient εdye and correction factor CF260nm for the dye spectrum at 260 nm wavelength into account.
    14. Store resulting strand at 4 °C under the exclusion of light
  5. Microscopy U2OS medium
    1. Prepare cell medium (0.5% FBS) as indicated:
      MEM                                                      48.75 ml
      Pyruvate                                                0.5 ml
      Penicillin/Streptavidin 100x               0.5 ml
      FBS                                                        0.25 ml
    2. Mix and filter medium under sterile conditions (hood)
    3. Store medium at 4 °C
  6. Microscopy sporozoite gliding medium
    1. Prepare sporozoite medium (RPMI + 6% BSA) always freshly as indicated:
      RPMI
      44.5 ml
      Penicillin-streptomycin
      2.5 ml
      Albumin fraction V
      3 g
    2. Dissolve albumin thoroughly
    3. Spin down unsolvable particles at 13k rpm for 5 min in the Heraeus Biofuge
  7. Click reaction mixture (CuAAC) (adapted from Hong et al., 2009)
    1. Prepare click reaction buffer as indicated:
      NaCl
      87.7 mg (cfinal: 150 mM)
      Na2HPO4
      14.7 mg (cfinal: 80.8 mM)
      KH2PO4
      25.9 mg (cfinal: 19 mM)
      Add 8 ml ultra-pure water
    2. Adjust the buffer to pH 7.4 by adding a few Microliter HCl (1 M)
    3. Add ultra-pure water to a total volume of 10 ml
    4. Dissolve aminoguanidine in ultra-pure water to a concentration of 100 mM
    5. Dissolve copper sulfate in ultra-pure water to a concentration of 20 mM
    6. Dissolve cRGD-alkyne in ultra-pure water to a concentration of 10 mM
      Note: Other tripeptides and ligands containing an alkyne function can be used equivalently. For azide-functionalized molecules the alkyne reaction partner is substituted and treated equally in this recipe.
    7. Dissolve sodium ascorbic acid in ultra-pure water to a concentration of 100 mM
    8. Dissolve THPTA in ultra-pure water to a concentration of 50 mM
    9. Premix 3 µl THPTA with 1.5 µl copper solutions thoroughly
    10. Mix 87.5 µl click reaction buffer with 1 µl cRGD-alkyne (cfinal: 0.1 µM), 1.5 µl THPTA/copper premix (cfinal: 0.5 µM/0.1 µM), 5 µl aminoguanidine (cfinal: 5 µM) and 5 µl ascorbate (cfinal: 5 µM)
    11. Store buffer at RT. Store aminoguanidine and copper solutions at 4 °C, peptide-alkyne and THPTA solutions at -20 °C. Prepare ascorbate solution always directly before use
  8. DNA annealing
    1. Dissolve DNA strands in ultra-pure water to concentration of 10 µM. In case of labeled top DNA strand, use the calculated values form the spectrophotometer measurement after resuspending. Store DNA strands at 4 °C, store hairpin strand at -20 °C
    2. For hairpin sensors, mix 3 µl top DNA (cfinal: 0.3 µM), 3 µl bottom DNA (cfinal: 0.3 µM) and 3.3 µl hairpin DNA (cfinal: 0.33 µM) to 70.3 µl NaCl buffer (1 M)
    3. Anneal the strands in thermal cycler. Program the thermal cycler: heat 10 min at 95 °C (denaturation), hold 4 s at 95 °C (annealing), repeat last step 399x while cooling down 0.2 °C at each step, final hold at 12 °C
    4. Add 20.4 µl additional bottom DNA (cfinal: 2.04 µM) to the annealed DNA

Acknowledgments

Thanks to all the coworkers from AG Frischknecht and AG Cavalcanti-Adam for enriching debates and suggestions.
    This work was funded by fellowships from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)-Projektnummer 240245660-SFB 1129, P1 and P15, and the Max Planck Society.

Competing interests

The authors declare that no competing financial interests exist.

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简介

[摘要 ] 组织内真核细胞或病原体的运动性是通过与其他细胞或细胞外基质特异性相互作用的转换来介导的。这些配体-受体粘附的生物物理特征有助于揭示驱动迁移的分子机制。牵引力显微镜或光学镊子通常用于测量细胞在基质上施加的细胞力。但是,牵引力显微镜的空间分辨率仅限于〜2 µm,使用光阱进行实验非常耗时。

在这里,我们介绍了仿生表面的生产,该表面能够通过合成配体实现特定的细胞粘附,同时通过使用分子张力传感器监控传递的力。将配体与双链DNA探针偶联,该探针具有确定的DNA解链力阈值。从而将pN范围内的受体介导力半定量转换为荧光信号,可以通过标准荧光显微镜在分辨率极限(〜0.2 µm)上检测到。

该测定的模块化设计允许改变所呈现的配体和DNA探针的机械强度,这为探测不同的真核细胞类型和病原体的粘附提供了多种可能性,此处以骨肉瘤细胞和伯氏疟原虫子孢子体为例。

[背景 ] 运动细胞和病原体以多种不同方式与环境相互作用(Parsons 等,2010; Nan ,2017; Muthinja 等,2018 )。例如,跨膜受体将单个细胞锚定在其环境中,并使其与其他细胞相互作用(Hynes ,1992)。整联蛋白是将细胞连接到细胞外基质的主要受体,它以双向方式传递力(Schoen et al。,2013)。一方面,细胞外机械信号被转导到细胞质中,在那里它们触发信号级联反应,从而控制各种细胞功能,如增殖,分化和迁移(Wang 等,1993; Janmey和Miller ,2011; Totaro 等。,2017 )。另一方面,由细胞骨架产生的力可施加到细胞外环境,引起组织形状改变并调节专门细胞中的细胞运动性(Iskratsch 等人,2014)。

为了测试细胞的整合素特异性粘附,在这里,我们第一次描述了一种“选项I”准备呈现环状基板精氨酸- 甘氨酸-天冬氨酸(的cRGD 同时通过聚乙二醇被阻止非特异性表面相互作用)配体(PEG )基钝化层(图1 A)。值得注意的是,通过改变配体,该方法可以容易地用于其他受体。

为了表征细胞力传递的不同方面,例如绝对张力值,方向或力的时空分布,存在几种方法,这些方法基于水凝胶的变形,微柱或分子的不老化,它们的空间分辨率和作用力范围不同(Polacheck and Chen,2016)。最新方法之一是通过基于荧光DNA的张力传感器可视化施加在单个配体上的牵引力(Wang和Ha ,2013; Blakely 等,2014; Zhang 等,2014)。这些双链(DS)DNA或发夹(特别是自退火单链(ss)DNA结构)的传感器被固定在体外表面上,并携带的配体(例如,的cRGD),使对受体(整合素) -特异性细胞互动。受力后,dsDNA区域可以展开,这可以用荧光传感器检测到(图1B)。结合标准的宽视野或全内反射显微镜,可以在高分辨率下确定力的时空分布,该分辨率仅受光学分辨率(〜0.2 µm)的限制。dsDNA的机械稳定性取决于其长度和GC含量(Zhang 等人,2015)。力F 1/2 时所需的展开50 的DNA传感器%,已在〜4之间以前单分子研究被校准- 60 PN用于改变DNA序列(Wang和哈,2013;张等人,2014 )。

在本文中所呈现的方法“选项Ⅱ”基础上的DNA发夹,其允许以量化的4范围内的单分子,力传递- 19对-N (张等人,2014;刘等人,2016) 。我们修饰了DNA链,以使各种配体能够以模块方式偶联,具体取决于目标受体。在封闭配置中,荧光标签非常靠近淬灭剂,因此是深色的。高于特定阈值力时,DNA 发夹解开并伸长,这降低了淬灭效率并产生了数字ON / OFF信号(图1B)(Woodside 等人,2006)。如果作用力不再存在,它们可以重新折叠(Bonnet 等,1998; Woodside 等,2006),这伴随着荧光的降低。这对于表征具有高迁移率的粘附形成(如运动性病原体)的细胞非常重要。在这里,DNA发夹传感器通过金纳米颗粒固定,金纳米颗粒用作额外的荧光猝灭剂。与其他传感器表面相比,这减少了由热力学波动以及光漂白引起的背景信号(Lang 等,2004; Liu 等,2016)。如果某个受体可以传递的力的范围先前未知,建议从最低的力探头开始,因为它定量地检测到高于其阈值的力。接下来,可以通过多路复用具有不同作用力阈值和不同荧光染料的传感器来扩展测定方法,以获得有关细胞作用力分布的半定量信息(Zhang 等人,2014)。

最后,我们展示了如何将“选项I”中确定的基底特定粘附力与分子张力传感器“选项II”结合起来,以进行牵引力测量,同时确保细胞对表面的适当粘附力。此方法“方案III” 可以防止小而能动的细胞(如疟原虫子孢子)频繁脱落,即使它们对与力传感器耦合的配体显示出较低的亲和力。

通过将分子张力传感器固定在这种适应性基质上,它们可用于细胞与细胞外配体粘附的广泛生物物理研究中。我们最近使用分子张力传感器来研究病原体(如病毒颗粒)与哺乳动物细胞之间的特异性粘附(Wiegand 等,2020)。在这里,我们提供了一个协议来研究不同细胞类型(包括疟原虫子孢子)与特定基质配体之间的相互作用。疟原虫的快速运动是感染周期的关键部分(Frischknecht和Matuschewski,2017),并已通过牵引力显微镜(Münter 等,2009)和激光镊子(Quadt 等,2016)进行了研究。请注意,分子传感器半定量检测通过单个受体传递的力的分布,尽管它们没有报告每个细胞传递的整体力(Goktas和Blank,2017),也没有报告没有集成其他荧光偏振技术(Brockman )的力方向等人,2018)。由于这些方法对实验过程的变化非常敏感,因此我们建议仔细控制方案的每个步骤。



D:\ Reformatting \ 2020-6-1 \ 2003095--1477蒂娜·韦根861625 \ Figs jpg \ Fig1 .jpg

图1. 体外DNA传感器力组件的实验设计和工作原理示意图。一。该协议中各个步骤的逐步表示,以生成配体呈现表面(选项I),具有基于DNA的力传感器的表面(选项II)以及在表面背景中显示的细胞粘附配体的组合以及带有另一种配体类型的基于DNA的力传感器(选项III)。简要地说,将玻璃盖玻片清洗干净,用PEG层钝化,然后将细胞配体,金纳米颗粒和DNA链固定在顶部。有关详细信息,指示的步骤将导致过程章节中的相应部分。乙。设计在轻松的构象和负载下DNA发夹力传感器。“发夹式” ssDNA(灰色)形成传感器的骨架,并与底部的金纳米颗粒和顶部的配体相连。顶部ssDNA(橙色)与荧光染料相连,底部ssDNA(蓝色)与淬灭剂相连。额外的底部ssDNA(绿色)增加了背景荧光的猝灭。DNA发夹探针的退火过程在R ecipe 8中进行了描述。施加力时,部分自退火的“发夹”链会通过破坏核苷酸之间的氢键而展开,从而降低两种染料之间的淬灭效率,从而局部增加在荧光信号中。图未按比例绘制。

关键字:分子力传感器, DNA发夹, 仿生表面, 受体介导力, 分子张力荧光显微镜, 疟原虫, 孢子体

材料和试剂


 


标准


1.5 ml塑料反应管(例如,Eppendo rf,目录号:0030125150)
铝箔
细胞罩
大小不同的锥形烧瓶
缓冲瓶
不同尺寸的玻璃烧杯
油浴用耐热玻璃盆
带吸头的不同大小的移液器
大小不同的Schlenk烧瓶
细尖镊金属,例如,#4,#7(西格玛,目录号:T6165)

超纯去离子水max。抵抗力18 。2M的Ω·cm的,例如,的MilliQ纯化
惰性气体(氩气)
氮气
Schlenk生产线(Schlenk烧瓶具有大开口,Schlenk烧瓶的盖子,真空和氮气的连接管)
d eionised(DI)水
 


消耗品


20 x 20 mm盖玻片#1(Carl Roth,目录号:H873.2)
DWK生命科学DURAN TM 过滤程序(第3号)(费希尔小号系统求解,目录号:09-841-090)
GE Healthcare公司的illustra TM ,NAP-5柱(费舍尔小号系统求解,目录号:10054394)
玻璃盖玻片架(自制)
Kimwipe纸巾(Kimberly-Clark Worldwide,目录号:05517)
分子筛3Å(Carl Roth,目录号:8487)
巴斯德移液器ISO 7712(230 mm)(Karl Hecht,目录号:40567002)
培养皿,PS,35/10 mm,带通风孔(Greiner Bio-One,目录号:627102)
无菌无菌CME旋转式注射器过滤器(Carl Roth,目录号:P818.1)
硅腔(Ibidi GmbH,目录号:80841)
铁氟龙盖玻片架(家用,可商购,例如,Thermo Fisher Scientific,目录号:C14784)
硅酮胶,Twinsil 22,加成固化的双硅酮(P icodent GmbH,目录号:13001000)
油浴用硅油(Sigma-Aldrich,目录号:85409)
 


化学制品


金纳米球,9 nm(nanoComposix ,自定义尺寸,9±2 nm,单宁酸中0.05 mg ml -1 )
(+)-L-抗坏血酸钠(Sigma-Aldrich,目录号:A7631)
3-(三乙氧基甲硅烷)propylisocyanate (Sigma-Aldrich公司,目录号:413364)
醋酸乙酯(Carl Roth,目录号:6784)
乙腈(用于HPLC的CHROMASOLV)(Honeywell International,目录号:60-002-31)
氨基胍盐酸盐(Sigma-Aldrich,目录号:A8835)
ɑ-硅烷-ω-炔PEG,(CH 3 O)3-Si-PEG(3000)-C≡CH(Rapp Polymere ,目录号:133000-70-71)
ɑ-硅烷-ω-叠氮基PEG,(CH 3 O)3-Si-PEG(3000)-N3(Rapp Polymere ,目录号:133000-5-71)
硫酸铜(Sigma-Aldrich,目录号:C1297)
乙醚(Sigma-Aldrich,目录号:309966)
二甲基亚砜(用于光谱)(DMSO)(默克(Merck),目录号:102950)
cRGD-炔烃(Biotrend Chemicals,定制合成:Cyclo (Arg - Gly -Asp-D- Phe - Pra ))
30%过氧化氢(默克(Merck),货号:107209)
LA- PEG(3400)-NH 2 (Biochempeg Scientific,目录号:HE039005-3.4K)
甲醇(卡尔·罗斯,目录号:0082)
N,N-二甲基甲酰胺(无水,99.8%)(DMF)(Sigma-Aldrich,目录号:227056)
氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
氢氧化钾(用于清洗浴)(Sigma-Aldrich,目录号:P5958)
磷酸二氢钾(KH 2 PO 4 )(Sigma-Aldrich,目录号:P5655)
QSY21(Thermo Fisher Scientific,Invitrogen TM ,目录号:Q20131)
硅烷-PEG- 甲氧基,Sil-PEG(2000)-m(Biochempeg Scientific,目录号:MF001020-2K)
碳酸钠(Na 2 CO 3 )(卡尔·罗斯,目录号:A135)
氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
磷酸氢二钠(Na 2 HPO 4 )(Sigma-Aldrich,目录号:255793)
StarRed-NHS(Abberior)(Abberior,目录号:1-0101-011-3)
硫酸96%(VWR CHEMICALS,目录号:1.08131.1000)             
甲苯(Merck,产品目录号:108325)
三乙胺(Sigma-Aldrich,目录号:471283)
醋酸三乙铵(TEAA)(Sigma-Aldrich,目录号:69372)
三(3-羟丙基三唑基甲基)胺(THPTA)(Sigma-Aldrich,目录号:762342)
单击反应混合物(CuAAC)(请参阅食谱)
干甲苯(请参阅食谱)
Sil-PEG(3500)-LA(请参阅食谱)
 


DNA链(由Integrated DNA Technologies,Inc . 定制合成)


注意:š 精灵退火部分下划线,顶链退火点缀,底链退火虚线; 有关文献中使用的发夹及其校准的列表,请参见Zhang等,2014和Liu等。,2016年。


顶部:5 ' -/ 5AmMC6 / CGC ATC TGT GCG GTA TTT CAC -3 '
底部NH 2- 淬火器:
5 ' -/ 5 ThioMC6-D / TTT GCT GGG CTA CGT GGC GCT CTT / 3AmMO / -3 '


发夹4.2 pN :
5'-/ 5AzideN / GTG AAA TAC CGC ACA GAT GCG TTT- GTA TAA ATG TTT TTT T CA TTT ATA C -TTT AAG AGC GCC ACG TAG CCC AGC AAA / 3ThioMC3-D / -3'


发夹12 pN: 
5'- / 5AzideN / GTG AAA TAC CGC ACA GAT GCG TTT- GGG TTA ACA TCT AGA TTC TA T TTT TTT TAG AAT CTA GAT GTT AAC CC -TTT AAG AGC GCC ACG TAG CCC AGC AAA / 3ThioMC3-D / -3 '
发夹19 pN: 
5'-/ 5叠氮化物N / GTG AAA TAC CGC ACA GAT GCG TTT- CGC CGC GGG CCG GCG CGC GG T TTT CCG CGC GCC GGC CCG CGG CG -TTT AAG AGC GCC ACG TAG CCC AGC AAA / 3ThioMC3-D / -3 '


标记的DNA链(请参阅食谱)
退火的DNA链(请参阅食谱)
 


酵素


的StemPro 的Accutase 细胞解离试剂(赛默飞世尔科技,Gibco公司® ,目录号:A1110501)
 





白蛋白组分V(Carl Roth,目录号:8076)
胎牛血清(美国)(Sigma-Aldrich,目录号:F2442)
MEMα(赛默飞世尔科技,Gibco公司® ,目录号:12561)
青霉素-链霉素(10,000单位/毫升)(赛默飞世尔科技,Gibco公司® ,目录号:15140148)
RPMI 1640培养基(赛默飞世尔科技,Gibco公司® ,目录号:11-875)
丙酮酸钠的100mM(100×)(赛默飞世尔科技,Gibco公司® ,目录号:11360070)
显微镜U2OS介质(请参阅食谱)
显微镜下的子孢子滑移介质(见食谱)
PBS缓冲液(请参阅食谱)
 


细胞系


伯氏疟原虫ANKA菌株(BEI资源(ATCC),目录号:MRA-871)
伯氏疟原虫NK65株,CS-GFP线(可选的使用,S EE 步骤H4)(未商购,从配合的寄生虫机构可请求;进一步的信息可参见纳塔拉詹。等人,2001 )
U2OS,骨肉瘤细胞(ATCC,目录号:HTB-96)
 


设备


 


300°C烤箱
冷冻室(-20°C和-80°C)
冰箱(4°C)
通风柜
离心机(Thermo Fisher Scientific ,型号:Heraeus Biofuge Pico)
离心机(Thermo Fisher Scientific ,型号:Heraeus Multifuge 1S-R;目录号:75004331)
热板配件(IKA ,型号:ETS-D5 ,目录号:0003378000)
HPLC(GE医疗保健小样大号IFE š ciences ,型号:ÄKTA P URE Ç hromatography 小号ystem)
HPLC色谱柱(Phenomenex ,型号:Luna 5u C18(2)-RP-HPLC ,目录号:00G -4252-N0)
Lyophilisator (LABCONCO ,型号:自由区加2.5升级联台式冷冻干燥系统,目录号:7670020)
电磁搅拌器(IKA ,型号:C-MAG HS 7 ,目录号:0003581200)
宽视场,倒置荧光显微镜(应用精密,型号:DeltaVision TM 精英中号icroscope 小号ystem,基于在Olympus IX71体)与自制环境室(购孵化例如,在OKOLAB)
显微镜滤光片组(适用精度,型号:DeltaVision TM Elite滤光片组)
显微镜物镜(奥林巴斯,型号:物镜60x / 1.4 NA PlanApo IX70)
Sonicator(EMAG Technologies ,型号:Emmi-H22 ,目录号:98217-DE)
分光光度计(NanoDrop Technologies ,型号:PEQLAB Nano Drop ND-1000)
热循环仪(Bio-Rad Laboratories ,型号:Bio-Rad T100 ,目录号:1861096)
 


软件


 


斐济只是使用ImageJ(版本1.52E; https://fiji.sc/)(Schindelin 。等人,2012)
堆对比度调整,插件斐济(Michálek 等人,200 7 )
程序


 


D:\ Reformatting \ 2020-6-1 \ 2003095--1477蒂娜·韦根861625 \ Figs jpg \ Fig2.jpg


图2 。实验程序概述。用镊子夹住玻璃盖玻片,将其插入特氟龙固定器(白色)中,然后将其移入装有食人鱼溶液的烧杯中进行清洁(步骤A)。为了使盖玻片钝化,将它们转移到玻璃固定器中,并与Schilk烧瓶中的Sil-PEG钝化溶液在油浴中于80°C孵育。可以将Schlenk烧瓶通过其阀门连接到真空和氮气管线上,以提供惰性反应容器(未显示,过程B)。清洁并干燥盖玻片后,通过在两张盖玻片之间孵育一滴相应溶液来固定特定的配体和金纳米颗粒(红色溶液)。为了再次去除彼此的盖玻片,请用移液器注入另外的水或PBS,并用镊子将上盖玻片提起(步骤s CE)。为了在倒置显微镜下观察表面,将它们用两种成分的硅胶(黄色和蓝色凝胶)粘合到培养皿中,培养皿的中心有一个定制孔(比盖玻片小2毫米)(步骤F)。Ť 他最终器件被示出在最后一个数字,其中介质和细胞可转移到显微镜之前加入。图片下方指示的步骤提供了更多信息。


 


清洁玻璃表面
在约100毫升新鲜制备的“ Piranha溶液” [ 3:1(v / v )硫酸:过氧化氢]中,在Teflon固定器中清洁并活化盖玻片1小时。确保盖玻片完全被溶液覆盖(图2,清洁)。搅拌食人鱼溶液,例如 ,用耐酸的巴斯德吸管。
注意:食人鱼溶液腐蚀性极强,并且会放热反应。不得与有机溶剂接触。在通风良好的通风橱中执行包含食人鱼溶液的每个工作步骤。


注意:从此处开始,仅用镊子处理盖玻片(图2)。


根据您所在机构的安全规定,小心地将其倒入适当的废物容器中,以除去盖玻片上的“食人鱼溶液”,例如,用水稀释10-20倍后将其丢弃。
用去离子水冲洗盖玻片3次,用超纯水冲洗两次。
在〜60瓦(标准超声处理功率)下,在超纯水中超声处理盖玻片一次,持续5分钟。
用镊子小心地保持氮气流中的表面干燥。
检查点:用眼观察,没有条纹或水珠保持否则重复小号TEP 小号A3- 一个4或放弃盖玻片。


 


玻璃表面钝化
将干燥的盖玻片转移到带有镊子的玻璃支架上,并避免在中心触摸它们。
在Schlenk烧瓶中,在连续的保护性氮气气氛下,按照表1所示制备钝化溶液(取决于所需的表面选项I - III,请参见图1A)(图2,钝化)。涉及有机溶剂的所有步骤(步骤B 2-B7)均应按照贵机构的安全规定在通风橱中进行,并保持通风。通过将Schlenk烧瓶连接到真空管线和氮气源来营造保护气氛。首先抽空烧瓶,然后用氮气冲洗并在随后减少氮气流,以添加粉末状PEG,干燥的甲苯和三乙胺。
 


表1. 三种不同类型表面的钝化成分(选项I -III)。当要测试细胞对特定配体的粘附力时,应选择选项I;选择II来获取此类特定配体的作用力;应选择配体对表面的特异性粘附力时,应选择选项III配体。


试剂


选择。我–


配体表面(请参阅步骤C)[绝对值。(摩尔比)]


选择。II –


发夹式力传感器(请参阅步骤D)[绝对值。(摩尔比)]


选择。III –


配体表面上的发夹力传感器(请参见步骤E)[abs。(摩尔比)]


Sil-PEG(2000)-米


 


6毫克(95%)


 


Sil-PEG(3000)-叠氮化物


6毫克(100%)


 


6毫克(95%)


Sil-PEG(3000)-炔


或6毫克(100%)


 


或6毫克(95%)


Sil-PEG(3500)-LA


 


0.35毫克(5%)


0.35毫克(5%)


干甲苯


20毫升


20毫升


20毫升


三乙胺


一滴(〜100 微升)


一滴(〜100 微升)


一滴(〜100 微升)


 


在保护气氛下,将盖玻片加入溶液中的Schlenk烧瓶中,然后关闭盖和气体供应。现在将反应保持在保护气氛下。
在78热管过夜- 80℃的油浴中。可以结合在磁力搅拌器上注满油的耐热玻璃桶来设置油浴。附加的恒温器可保持温度恒定。
在玻璃固定器中用乙酸乙酯超声处理5分钟,以钝化盖玻片。
在玻璃固定器中,用甲醇超声清洗钝化的盖玻片,再清洗5分钟。
暂停点:如果钝化玻璃表面不立即使用,则最多可以在甲醇中保存5天。


用镊子小心地保持氮气流中的表面干燥。氮气流可以用压缩空气枪小心地添加。确保在整个过程中不要划伤钝化的表面。
检查点:甲S代表适当钝化控制添加染料(例如,100 nM的加星号的在PBS中)或在相对于钝化的非钝化盖玻片并在显微镜下观察其附接至表面种子粘着细胞。


要重新使用,请在氢氧化钾浴中将用过的Schlenk烧瓶和玻璃样品架从PEG残留物中清洗过夜,然后用去离子水洗涤。
 


选项I – 固定特定细胞配体
准备100 µl 反应混合物,以进行含有所需细胞配体的铜催化的叠氮化物-炔烃环加成反应(CuAAC,请参见配方7)。
注意:分别为叠氮化物或炔配体选择合适的炔或叠氮化物官能化PEG。


准备一个带有底膜的湿气室,在一个培养皿中放置一块盖玻片的湿纸巾内,放置一个湿纸巾(图2,固定)。Parafilm用于将盖玻片固定在其位置,并在以后简化“夹心”玻璃的分离。
将一滴100 µl滴答混合物放在钝化并干燥的盖玻片上。
将第二张相同的盖玻片放在第一个盖玻片上。
在室温(RT)下将“三明治”孵育2小时。
 


方案二– 固定DNA发夹力传感器
放置250的下降- 将300μl上的钝化和干燥盖玻片顶部的金纳米颗粒的溶液。
在室温下孵育盖玻片30分钟。
同时,使用“顶部”,“底部”和“发夹” ssDNA杂交发夹力传感器。按照指示使用DNA混合物和退火程序(请参见第8条)。如果购买的DNA链未经标记,则应在上一步中将DNA和荧光团偶联(有关下面标记DNA链的信息,请参见第4条)。
通过在玻璃杯之间小心吸取300 µl超纯水来分离表面,并用镊子将它们捡起(图2,固定)。通过用〜10 ml超纯水彻底冲洗,将其从上清液中的金纳米颗粒溶液中清除。
检查要点:通过盖玻片的粉红色光泽确认金纳米颗粒已成功固定。


除去盖玻片边缘上有薄纸的多余液体,不要让它们完全干燥或接触它们。
将一滴100 µl DNA混合物滴在镀金的盖玻片上。
放置第二张相同的盖玻片,金纳米颗粒涂层的一面朝下放在第一张盖玻片上。
在排除光线的情况下,将“三明治”在4°C下孵育过夜。
准备含有三肽/配体的100 µl 用于CuAAC 的反应混合物(请参见第7条)。
通过用PBS洗涤,仔细地从DNA混合物中分离并清洁表面(类似于步骤D4)。
除去盖玻片边缘上有薄纸的多余液体,不要让它们完全干燥或接触它们。
将一滴10 0 µl单击混合物放在DNA包被的盖玻片上。
放置第二张相同的盖玻片,将DNA涂层的一面朝下放在第一张盖玻片上。
在避光条件下,在湿度室中于室温下将“三明治”孵育2小时,以避免荧光团漂白。
 


选项III – 特定细胞配体和DNA发夹力传感器的组合
准备100 µl 含1st 配体的CuAAC的1st 反应混合物(请参见第7条)。
准备一个在培养皿内有湿纸巾的湿气室,并在其底部放上盖玻片的底部放平膜。
将一滴100 µl滴答混合物放在钝化并干燥的盖玻片上。
将第二张相同的盖玻片放在第一个盖玻片上。
在室温下将“三明治”孵育2小时。
通过用PBS洗涤(与步骤D4相似),仔细地从点击混合物中分离并清洁表面。
进一步的步骤是等于“d)的DNA发夹力传感器固定化固定化” 小号TEP 小号使用2 D1-D14 第二配体在小号TEP D9。
 


显微镜样品制备
通过用PBS洗涤(与步骤D4相似),仔细地从点击混合物中分离并清洁表面。
除去盖玻片边缘上有薄纸的多余液体,不要让它们完全干燥或接触它们。
将盖玻片涂胶,使功能化表面朝上进入培养皿,底部带有定制孔(比盖玻片小2 mm)。因此,将约200 µl的Twinsil 22硅1:1(例如)与移液器吸头混合,然后将胶水涂在孔的边缘,例如盖玻片完全覆盖孔并被胶水包围。
等待胶水的固化时间(〜5分钟,检查剩余的胶水)。如有必要,通过用PBS清洗表面以清除胶水污染。通过分别比较仅钝化盖玻片与固定化配体(步骤C)或力传感器(步骤D)的细胞附着(程序G)和均质背景荧光,
检查细胞配体和力传感器是否成功固定。
 


细胞分析(U2OS)
将用于观察细胞的腔室置于37°C和5%CO 2 气氛中。
在步骤F中制备的覆盖功能化盖玻片的陪替氏培养皿中填充2-3 ml细胞培养基(0.5%FBS)(请参阅配方5)。使用低FBS含量的培养基以防止非特异性配体相互作用并改善显微镜。使用细胞培养通风橱以防止培养基或细胞培养物污染。
按照标准规程(例如,基本细胞培养规程(Helgason和Miller ,2004)),用PBS洗涤从培养瓶中收获贴壁细胞,并用Accutase分离。
注意:Accutase可以使细胞轻柔地分离出来,从而获得更快的铺展效果。


稀释浓度的种子细胞可以不受干扰地扩散。在U2OS 10的情况下,5个细胞达到足够的密度(从T-25和35 mm的培养皿表面以大约1:10的稀释度计算)。
等到细胞沉淀下来并开始在表面上高效繁殖(10-30分钟)。
注意:力传递最好在接种细胞后30分钟观察到,并显示为打开的DNA发夹传感器的亮点和放射状条纹(图3)。


 


D:\ Reformatting \ 2020-6-1 \ 2003095--1477蒂娜·韦根861625 \ Figs jpg \ Fig3.jpg


图3. U2OS 细胞在特定配体呈现表面上的粘附力和施加力。用(A)选项I:cRGD功能化和(B)选项II:cRGD连接的DNA播种在表面后15-20分钟内的U2OS细胞的代表性明场,干涉反射显微镜(IRM)和荧光宽视场图像发夹传感器(4.2 pN打开力;StarRed荧光团)。比例尺= 10 µm。


 


子孢子测定
尝试使盖子的边缘尽可能干燥,而功能化表面的中心应保持潮湿。这简化了硅腔室的建立。
按压硅胶腔室,使其粘性侧在功能化盖玻片的边缘,以最大程度地减少细胞分布区域(注意:硅胶棒最好放在干玻璃上)。购买的多孔室可以切成合适的尺寸。可以跳过此步骤,但是易于生成足够的寄生虫密度。
将15 µl子孢子培养基(6%BSA)(参见配方6)填充到硅腔中。
加入15 µl冰冷的PBS,其中含有来自6-8对感染良好的蚊子唾液腺的新鲜分离的子孢子(有关蚊子感染和子孢子的详细收集方法,请参见Prinz 等人,2017),得到3%-BSA-培养基。
笔记:


也可以使用减少FBS的细胞培养基(0.5%FBS)。
不要将唾液腺长时间放置在冰上,因为这可能会损害子孢子的活力。
GFP标记的子孢子的使用简化了感染良好的唾液腺的选择及其分离。它们可以在配体呈递的表面可以采用(选项I,参见图ü 重新4 甲),但必须小心以力传感器(图一起使用它们时选择合适的滤光器组URE 4 乙)吨o避免荧光漂白剂通过。
在2,000 xg 的离心机中离心3分钟将细胞旋转至表面。有充分根据的硅室可以承受此程序。
附着在表面约30分钟后即可直接观察到运动性(图4)。
D:\ Reformatting \ 2020-6-1 \ 2003095--1477蒂娜·韦根861625 \ Figs jpg \ Fig4.jpg


图4。子孢子(伯氏疟原虫)在特定配体存在的表面上滑动。(A)表达GFP的子孢子播种在表面上5分钟后的代表性明场,干涉反射显微镜(IRM)和荧光广域时移图像,在选项I播种后的5分钟后是cRGD-功能化和(B)WT子孢子在选项I上用选项II播种在表面:cRGD连锁的DNA发夹传感器(4.2 pN打开力; StarRed-荧光团)。对于选项II骨肉瘤细胞(空心箭头)与子孢子作为用于基于DNA的力传感器的阳性对照一起接种(见图URE 3)。子孢子粘附并在两个表面上滑动,但是,DNA发夹传感器未检测到张力信号。这可能是由于亚阈值力,很少的表面相互作用或太短的粘附时间(子孢子以〜2 µm / s的速度移动)而导致张力探针无法检测到展开,而粘附在同一基质上的骨肉瘤细胞发出荧光信号(空心箭头)。明亮的荧光点(全箭头)是从蚊子唾液腺中纯化子孢子后产生的自身荧光残留物。比例尺= 10 µm。


 


显微镜设置
在配备有针对细胞条件(37°C; 5%CO 2 )的定制环境室的荧光广角显微镜(Olympus)上进行成像。子孢子通常在不提供CO 2的情况下在室温下成像,但我们也成功地在细胞条件下进行了滑行运动分析。
用偏光滤光片组拍摄明场图像。
用激发带通滤光片632/22 nm和发射滤光片679/34 nm拍摄荧光显微镜图像。
使用激发带通滤光片542/27 nm以IRM模式(设置发射带通滤光片525/48 nm)以最小的曝光时间和强度拍摄干涉反射显微镜(IRM)图像。因此,IRM观察可用作成像过程中细胞粘附于表面的低漂白控制。
 


数据分析


 


不含力传感器的受体表面(选项I)可用于对在非粘附性PEG化的表面上已定义的配体的细胞粘附特性进行定性测试。明场成像或IRM可以验证细胞是否紧密粘附在表面上,例如这种方法不需要荧光显微镜检查。


  此外,可以对细胞在表面上的沉降和扩散时间进行定量分析(数据分析请参见Schaufler 等人,2016),对于活动子孢子则可以使用滑移运动的定量分析(数据分析请参见Prinz 等人,2017)。用于不同细胞/寄生虫系或不同配体之间的可比数据分析。


  DNA力传感器表面(选项II 和III)以特定的阈值力提供数字ON-OFF信号。传感器表面的荧光显微镜图像可提供有关所施加力的空间,时间和半定量信息,这些信息由荧光信号的瞬时出现(发夹)指示。因此,首先使用Fiji软件调整荧光显微镜图像的亮度和对比度,以更好地观察信号。对于延时成像,使用“堆栈对比度调整”插件来校正光漂白。可以从反映dsDNA打开的相对量的局部亮度中生成半定量力读数,并通过颜色编码的LUT(例如“光谱”)进行显示(有关更多详细信息,请参见Zhang 等人,2014年,补充图7)。但是,这种定量分析受到光学分辨率的限制,因为通常每个衍射限制点将包含多个传感器。


  为了缩小绝对牵引力值,可以通过改变DNA序列,使用具有不同刚度的DNA传感器重复该实验。可以将多个与不同荧光团偶联的传感器组合在一个表面上,以获得多重结果(参见Zhang 等人,2014)。


 


笔记


 


准备适当体积的等分试样用于冷冻培养基或溶液,以避免重复的冻融循环并延长其保质期。
用移液管小心地在三明治内添加300 µl PBS,从反应混合物中分离并清洁表面。随后提起顶部盖玻片,并用PBS洗涤。确保在整个过程中不要刮擦表面。
避免在任何时候使表面变干,因为回缩的液体会由于表面上的表面张力而产生伪影。
具有NHS功能的其他荧光染料和猝灭剂可用于标记DNA,具体取决于显微镜中滤光片的可用性。考虑具有高量子产率和接近金纳米颗粒的粒子共振峰的发射波长的染料,以实现更强的猝灭(Kang 等,2011)。请注意,细胞的自发荧光,尤其是来自细胞核的自发荧光,可以与来自细胞下方的传感器信号重叠。对于具有高核体比的较小细胞(例如,疟原虫子孢子),像StarRed这样的远红色染料可以使这种信号自发荧光干扰最小化。
 


菜谱


 


PBS缓冲液
按照指示制备PBS:
氯化钠8.006克(终浓度:137毫米)             


KH 2 PO 4 1.375 g(最终浓度:10.1 mM)             


氯化钾201.3毫克(终浓度:2.7毫米)             


Na 2 HPO 4 255.5 mg(最终浓度:1.8 mM)             


添加0.8升的超纯净水
通过加入几毫升HCl(1 M)将pH值调节至7.4
加超纯净水至总体积为1 L
将缓冲区存储在RT
Sil-PEG(3500)-LA
在一个小的Schlenk 烧瓶中,在氮气大气压下用磁力搅拌棒将145 µmol NH 2 - PEG(3400)-LA溶于3 ml DMF中
加入159.5 µmol 3-(三甲氧基甲硅烷基)丙基异氰酸酯(1.1 分子倍数PEG)
STI - [R在室温下过夜
将溶液冷却至4°C
甲DD冷却乙醚1个小时
用冷却的乙醚在玻璃粉(3号)上洗涤反应物
在真空下将所得的Sil-PEG-LA干燥至干燥
将粉末在惰性气体下储存在-20°C
干甲苯
在烤箱中充分干燥分子筛
用真空吸尘器倒空锥形瓶,并添加氮气气氛
在保护气氛下添加甲苯,并正确关闭烧瓶
等待数小时以清除甲苯中过多的水
在氮气氛下室温保存
大号阿贝尔荷兰国际集团的DNA链的
将超纯水中的DNA 溶解至1 mM的浓度
将染料-NHS分别浸入干燥的DMSO中至10 mM
将105.99 mg Na 2 CO 3 溶解在1 ml超纯水中
在150 µl PBS中混合60 µl溶解的DNA上链(c final :0.2 mM),60 µl 溶解的染料-NHS(c final :2 mM)和30 µl Na 2 CO 3 溶液(c final :0.1 mM)。用DNA bot tom链和淬灭剂-NHS进行相同的处理
调节pH至8.5
排除光线,在室温下搅拌过夜
按照制造商用户手册中的说明在NAP-5柱中纯化混合物
用HPLC(梯度高压液相色谱)纯化混合物。使用Luna 5u C18(2)-RP-HPLC色谱柱,流动相为醋酸三乙铵(TEAA)(0.1 M)和乙腈,其90分钟内的浓度为10%至50 %,20分钟内的浓度为50%至90%
收集同时显示DNA(260 nm)和染料在相同时间的两个最大吸收峰的馏分
将产品冷冻至-80°C,然后冻干以除去溶剂
在超纯水中重悬纯净产品
确定的与使用NanoDrop分光光度计测定浓度的吸光度甲DNA; 1mm 的DNA在260 nm 和吸光度A 染料; 在各染料/猝灭剂的激发波长下为1mm
通过以下三种方式测量浓度:
 










 


注意:考虑染料在260 nm波长处的消光系数和校正因子CF 260 nm 。


将所得的链条在避光条件下储存在4 °C
显微镜U2OS介质
按照指示准备细胞培养基(0.5%FBS):
MEM 48.75毫升             


丙酮酸0.5毫升             


青霉素/ 链霉亲和素100x 0.5毫升             


FBS 0.25毫升             


在无菌条件下(通风橱)混合并过滤培养基
将介质储存在4°C
显微镜下子孢子滑移介质
按照指示始终新鲜准备子孢子培养基(RPMI + 6%BSA):
RPMI 44.5毫升             


青霉素链霉素2.5毫升             


白蛋白级分V 3 g             


彻底溶解白蛋白
在Heraeus Biofuge中以13k rpm f 或5分钟旋转不可溶解的颗粒
点击反应混合物(CuAAC)(改编自Hong 等,2009)
                                                                                                            制备点击反应缓冲所示:
氯化钠87.7毫克(C 最终:150毫摩尔)的Na 2 HPO 4 114.7毫克(C 最终:80.8毫摩尔)KH 2 PO 4 25.9毫克(C 最终:19毫摩尔)甲DD8毫升超纯水             
             
             
                      通过添加一些微升HCl(1 M)将缓冲液的pH值调节至7.4
加入超纯净水至总体积为10毫升
在超纯水中溶解氨基胍至100 mM的浓度
将硫酸铜在超纯水中溶解至20 mM的浓度
溶解在超纯WA的cRGD炔吨ER至10mM的浓度
注意:可以等效地使用其他具有炔烃功能的三肽和配体。对于叠氮化物官能化的分子,炔烃反应伙伴在该配方中被取代并均等处理。


溶解钠ascorb 在超纯笏IC酸ER至100mM的浓度
将THPTA在超纯水中溶解至50 mM的浓度
预混物3微升THPTA用1.5微升共PPER溶液thoroughl ÿ
将87.5 µl点击反应缓冲液与1 µl cRGD-炔烃(最终浓度:0.1 µM),1.5 µl THPTA /铜预混液(最终浓度:0.5 µM / 0.1 µM),5 µl氨基胍(最终浓度:5 µM)和5 µl混合抗坏血酸(最终浓度:5 µM)
将缓冲区存储在RT。将氨基胍和铜溶液储存在4°C,将肽炔和THPTA溶液储存在-20°C。使用前总是直接准备抗坏血酸溶液
DNA退火
将DNA链溶于超纯水中,浓度为10 µM。如果标记了顶部DNA链,请在重悬后使用分光光度计测量值计算得出的值。将DNA链存储在4°C,将发夹链存储在-20°C
对于发夹型传感器,将3 µl顶部DNA(c final :0.3 µM),3 µl底部DNA(c final :0.3 µM)和3.3 µl发夹DNA(c final :0.33 µM)混合到70 .3 µl NaCl缓冲液(1 M )
在热循环仪中对绞线进行退火。对热循环仪进行编程:在95°C加热10分钟(变性),在95°C保持4秒钟(退火),重复最后一步399x,同时每步冷却0.2°C ,最后保持在12°C
向退火的DNA中添加20.4 µl额外的底部DNA(最终浓度:2.04 µM)
致谢


 


感谢AG Frischknecht 和AG Cavalcanti-Adam的所有同事,丰富了辩论和建议。


  这项工作是由来自德意志奖学金资助的研究联合会- (DFG,德国研究基金会)Projektnummer 240245660-SFB 1129,P1和P15,以及马普学会。


 


利益争夺


 


作者宣称不存在任何竞争性的经济利益。


 


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引用:Wack, M., Wiegand, T., Frischknecht, F. and Cavalcanti-Adam, E. A. (2020). An in vitro DNA Sensor-based Assay to Measure Receptor-specific Adhesion Forces of Eukaryotic Cells and Pathogens. Bio-protocol 10(17): e3733. DOI: 10.21769/BioProtoc.3733.
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