参见作者原研究论文

本实验方案简略版
Jul 2021
Advertisement

本文章节


 

Characterization of the Elasticity of CD4+ T Cells: An Approach Based on Peak Force Quantitative Nanomechanical Mapping
CD4+T细胞弹性的特征:一个基于峰值力定量纳米力学图的方法   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

CD4+ T cells are essential players in orchestrating the specific immune response against intracellular pathogens, and in inhibiting tumor development in an early stage. The activation of T cells is triggered by engagement of T cell receptors (TCRs). Here, CD3 and CD28 molecules are key factors, (co)stimulating signaling pathways essential for activation and proliferation of CD4+ T cells. T cell activation induces the formation of a tight mechanical bond between T cell and target cell, the so-called immunological synapse (IS). Due to this, mechanical cell properties, including stiffness, play a significant role in modulating cell functions. In the past, many approaches were made to investigate mechanical properties of immune cells, including micropipette aspiration, microplate-based rheometry, techniques based on deformation during cytometry, or the use of optical tweezers. However, the stiffness of T lymphocytes at a subcellular level at the IS still remains largely elusive.


With this protocol, we introduce a method based on atomic force microscopy (AFM), to investigate the local cellular stiffness of T cells on functionalized glass/Polydimethylsiloxan (PDMS) surfaces, which mimicks focal stimulation of target cells inducing IS formation by T cells. By applying the peak force nanomechanical mapping (QNM) technique, cellular surface structures and the local stiffness are determined simultaneously, with a resolution of approximately 60 nm. This protocol can be easily adapted to investigate the mechanical impact of numerous factors influencing IS formation and T cell activation.


Graphical abstract:




Overview of the experimental workflow.

Individual experimental steps are shown on the left, hands on and incubation times for each step are shown right.


Keywords: CD4+ T cell (CD4+ T细胞), AFM (AFM), Stiffness (僵硬度), Elasticity mapping (弹性图谱), Peak Force QNM (峰值力QNM)

Background

T cells belong to the adaptive immune system and can be classified as CD4+ T cells or CD8+ T cells. CD4+ T cells are essential for orchestrating the immune responses, and CD8+ T cells, also known as cytotoxic T lymphocytes, are the key players in eliminating tumor and pathogen-infected cells (Taniuchi, 2018). T cells are activated by the engagement of T-cell receptors (TCRs) with the matching antigen. Consequently, CD3 molecules, one key component of the TCR complex, transduce the signal to activate downstream pathways, leading to the formation of a tight junction between T cells and target cells, termed the immunological synapse (IS). In addition, engagement of co-stimulatory factor CD28, which triggers the production of interleukins (e.g., IL-6), initiates signaling pathways essential for sustainable activation and proliferation (Esensten et al., 2016). In addition to a complex ballet of receptor-ligand bonds, a fundamental rearrangement of the cytoskeleton takes place during IS formation, including the reorientation of the microtubule-organizing center (MTOC) towards the IS, and the formation of an F-actin ring structure at the IS. In between, the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1) binds its ligand on the target cells, to seal and stabilize the IS (Bromley et al., 2001). Upon T cell activation, the intracellular concentration of Ca2+ ions is drastically enhanced via Ca2+ influx (Friedmann et al., 2019). Ca2+ serves as an essential second messenger in T cells to regulate their activation, proliferation, and effector functions (Trebak and Kinet, 2019).


Mechanical properties, including stiffness and mechanical forces, play a significant role in modulation of cell functions, including those of T cells (Jansen et al., 2015). A recent study has demonstrated that cytotoxic T lymphocytes optimize their killing function by applying a mechanical force (Basu et al., 2016). T cells are able to generate mechanical forces (pushing and pulling forces) in the piconewton (pN) range upon activation (Husson et al., 2011; Hu and Butte, 2016). This force generation requires a sustained elevation of intracellular calcium, as well as integrity of the functional F-actin network, and phosphoinositide 3-kinases (PI3K) signaling (Hui et al., 2015; Basu et al., 2016). Other work shows that activation of T cells requires mechanical forces (Hu and Butte, 2016), and that T cells respond to environmental stiffness cues (Majedi et al., 2020). Changes in cell stiffness can regulate force generation (Harrison et al., 2019). However, the stiffness of T lymphocytes at a subcellular level at the IS still remains largely elusive.


Over the last 20 years, many different approaches were used to address the stiffness and stiffening processes in eukaryotic cells, especially immune cells. Early approaches, such as micropipette aspiration, and microplate-based rheometry, involved complex technical structures, in which the cells were inserted or incorporated and mechanically stressed (Hochmuth, 2000; Desprat et al., 2005). Cytometry-related approaches are based on principles like deformation during deceleration in flow, or twisting triggered by membrane-bound metal beads in a magnetic field. Those methods are limited to a constant fluid stream to operate. In contrast, methods which are able to characterize elastic properties of cells in an environment mimicking physiological conditions, like cell monolayers and cell-cell interactions, are capable of providing much deeper insights into biomechanical processes. Optical tweezers use focused laser beams to generate attractive or repulsive forces in the pN range, and are considered a promising tool to characterize and modify biomaterials. Hence, this method is mostly used to move or sort cells, and is still not broadly applied for the characterization of local cell properties (Killian et al., 2018).


Atomic force microscopy (AFM) is another biophysical method that can be used to determine cell forces in the pN range. AFM applies a mechanical probe, often made of silicon or silicon nitride with a nanometer-thin tip, that is moved along the cell surface, and interacts with each point of a defined scanning grid. Hence, it enables the examination of height profiles, or measures mechanical properties with spatial and force resolutions at the nanometer and pN ranges, respectively (Loskill et al., 2014; Thewes et al., 2015). In recent years, AFM has evolved as one of the most important tools in cell biology to study adhesive forces, and cell properties, such as deformation and elasticity (Scheuring and Dufrêne, 2010; Pi and Cai, 2019).


Recently, we established a peak force quantitative nanomechanical mapping-based method, for simultaneously determining the surface profile and stiffness of live T cells during IS formation at a subcellular level, with a resolution of ~60 nm (Jung et al., 2021). In this method, antibodies against CD3, CD28, and LFA-1 are immobilized on substrates, mimicking the focal stimulation of target cells that would induce IS formation in T cells. This protocol can be easily adapted to investigate the mechanical impact of a broad spectrum of intracellular or extracellular factors influencing IS formation and T cell activation.

Materials and Reagents

  1. Cell culture flasks 25 mL (Sarstedt, catalog number: 83.3910.502)

  2. Glass slides (Carl Roth, catalog number: H872.1)

  3. Glass cover slips 25 mm (VWR, catalog number: 631-1584)

  4. Falcon® 24-well plates (Corning, catalog number: 353047)

  5. Scalpel blades (B. Braun Melsungen, catalog number: 5518075)

  6. Fluorodish Petri dish 50 mm (World Precision Instruments, catalog number: FD35-100)

  7. Polypropylene tubes 15 mL (Fisher Scientific, catalog number: 11507411)

  8. Polypropylene tubes 50 mL (Fisher Scientific, catalog number: 10788561)

  9. MLCT AFM probes (Bruker AFM Probes, catalog number: MLCT-10)

  10. Lymphocyte separation media 1077 (PromoCell GmbH, catalog number: C-44010)

  11. Hanks’ balanced salt solution (Sigma-Aldrich, catalog number: H6648)

  12. CD4+ T Cell Isolation Kit human (Miltenyi, catalog number: 130-096-533)

  13. AIM V cell culture medium (Thermo Fisher Scientific, Gibco, catalog number: 31035025)

  14. Fetal bovine serum (FBS) (Thermo Fisher, Gibco, catalog number: A4766801)

  15. Phosphate-buffered saline (PBS) (Thermo Fisher, Gibco, catalogue number: 10010056)

  16. Polyornithine (Sigma-Aldrich, Merck, catalog number: MFCD00286305)

  17. anti-LFA-1 (ITGAL) antibody, mouse monoclonal (Antibodies-Online, catalog number: ABIN135680)

  18. mouse anti-human CD28 antibody, mouse monoclonal (BD Pharmingen, catalog number: 555725)

  19. mouse anti-human CD3 antibody, mouse monoclonal (Diaclone, catalog number: 854.010.000)

  20. Sylgard 184 Silicone Elastomer Kit (Dow Europe GmbH, catalog number: 1317318)

  21. For alternative cantilevers (see Notes): qp-BioT/qp-BioAC (Nanoandmore GmbH, catalog numbers: qp-BioT-20/qp-BioAC-20)

Equipment

  1. CO2 incubator (Heracell Vios 160i, Thermo Fisher Scientific, catalog number: 51030403)

  2. Centrifuge (Hettich Universal 32R, with 50 mL fixed angle rotor)

  3. Centrifuge (Eppendorf, model: 5424, with 2 mL fixed angle rotor)

  4. AutoMACS Pro Separator (Miltenyi Biotec, catalog number: 130-092-545)

  5. Cell and Particle Counter (Beckman Coulter Z2, catalog number: 8043-30-0016)

  6. Electrostatic deionizer (Eltex Elektrostatik GmbH, catalog number: W0150L025U99)

  7. Stove for 60°C PDMS curing (Memmert, model: 100-800)

  8. Diaphragm laboratory pump (KNF, catalog number: N811KN.18)

  9. Atomic Force Microscope (Bruker Corp., model: BioScope Catalyst)

  10. Bruker calibration table for laser alignment (Bruker Corp., model: Easy Align)

  11. Fluorescence microscope (Leica Microsystems, model: DMI 4000 B)

  12. Tweezers (e.g., Agar Scientific, catalog number: AG5596-TI)

  13. Cantilever Holder (for measurements in fluids) (Bruker Corp., model: CAT-PCH)

Software

  1. Research NanoScope, AFM operation software (Bruker Corp., version 9.1, 119071)

  2. NanoScope Analysis software (Bruker Corp., version 1.8, 132257)

  3. Excel 2016 (Microsoft)

  4. GraphPad Prism 6 (GraphPad)

Procedure

  1. Isolation and culture of CD4+ T cells

    1. Prepare peripheral blood mononuclear cells (PBMCs) from healthy donors, as previously described (Kummerow et al., 2014). Briefly, for separation of mononuclear cells from human blood, we used lymphocyte isolation medium, according to the manufacturer's instructions, and carry out the density gradient centrifugation at 450 g (acceleration: 1, deceleration: 0) and room temperature (RT) for 30 min. PBMCs were collected, and remaining erythrocytes were lysed with 1–3 mL of erythrocyte lysis buffer (see Recipes) for 1–2 min. Estimated duration: 1.5 h.

    2. Negatively isolate human CD4+ T cells from PBMCs, using the AutoMACS Pro Separator with the CD4+ T cell Isolation Kit human, according to manufacturer’s protocols. Estimated duration: 1 h.

    3. Resuspend isolated CD4+ T cells at a density of 3 × 106/mL, in a 24-well plate with AIM V medium with 10% fetal bovine serum (FBS) (1 mL/well), and keep the plate at 37°C with 5% CO2 for 24 h.

    4. Prior to AFM experiments, measure the cell density with an automated cell counter, remove the media by centrifugation (1,200 rpm at RT for 5 min) and adjust the cell suspension to a density of 2 × 105–3 × 105/mL, by resuspending the cells in AIM V medium without FBS.


  2. Preparation of antibody-functionalized coverslips (see Video 1)

    1. Clean glass coverslips, by wiping with 70% ethanol.

    2. With a permanent marker, mark a center spot with a diameter of approximately 7 mm at the backside of the glass coverslip.

    3. Coat the center spot with 40 µL of Polyornithine solution (optimized concentration for T cells: 100 µg/mL, in sterilized ddH2O) at RT for 30 min.

    4. Remove the polyornithine solution with a diaphragm laboratory pump and leave the coverslip to air dry for 10 min.

    5. Apply 20 µL of antibody dilution (in PBS) and incubate at 37°C for 30 min, to functionalize a spot of the glass coverslip. Store at 4°C overnight. In our experiments, the following combination of antibody concentrations showed the best results for IS formation (optimal concentrations might differ if other factors are investigated):

      1. αLFA-1 (9 µg/mL)

      2. αCD-3 (30 µg/mL)

      3. αCD28 (90 µg/mL)


        Video 1. Sample preparation.


  3. Preparation of PDMS, and antibody functionalization of PDMS surfaces

    1. Slowly pass all materials (e.g., Petri dishes, spatulas) through the arch of an electrostatic deionizer (~2 s), to remove any static charges before bringing them into contact with Polydimethylsiloxane (PDMS) elastomer components A (methylhydrosiloxane-dimethylsolioxane) and B (vinyl-terminated polymethylsiloxane).

    2. Mix component A and B in a 10:1 ratio in polypropylene tubes, to create PDMS with a stiffness of approximately 2.5 MPa (adapt according to the manufactures’ protocol, if other stiffnesses are addressed)

      Note: When handling the individual PDMS components, strictly use polypropylene materials (e.g., VWR Graduated Polypropylene Beaker 100 mL, catalog number 213-3918; Sarstedt Stirring Rod 120 mm, PP, catalog number 81.970), and Kimtech G3 Sterling Nitrile gloves (e.g., VWR catalog number 112-4879).

    3. Pour the mixture into 50-mm flat bottom Petri dishes (e.g., Fluordish), and make sure to cover the surface with a layer of approximately 1.5 mm. Leave to settle for 1 h.

    4. Place in an incubator to cure at 60°C for 16 h.

    5. Prior to AFM measurements, cut 10 × 10 mm squares from the PDMS layer with a scalpel blade, turn the PDMS piece over, and continue with the bottom side.

    6. Continue the functionalization process like described for glass cover slips (B), and apply cells as described below (D).


  4. Application of T cells on antibody functionalized surfaces (see Video 1)

    1. Pipette 100 µL of CD4+ T cell suspension in AIM V medium (without FBS) into the marked center spot of the functionalized surface (Figure 1A, approximately 2 × 104–3 × 104 will be transferred).

    2. Allow the cells to settle and interact with the functionalized surface in a wet chamber at 37°C and 5% CO2 for 15 min (plastic Petri dish lined with wet paper tissue; Figures 1A–1B), to avoid drying.

    3. Mount the functionalized glass cover slip onto a glass slide. Pipette 2 µL of sterilized ddH2O between the glass layers, to make sure that the functionalized cover slip or PDMS piece is held by cohesion, and will not move during the measurements (Figures 1C–1D).

    4. The sample should be used immediately. Discard the sample and freshly prepare a new one after 60 min of measurements, since the cells will die and start detaching from the surface at RT and without increased partial CO2 pressure.


  5. AFM preparation and calibration

    Note: In our experiments, the NanoScope 9.1 software was used to operate a BioScope Catalyst system (Bruker, Santa Barbara, USA) mounted onto a Leica DMI 4000 B optical microscope. All methods are specifically described for these systems, but the procedures can be adapted for other software versions and AFM systems, allowing peak force tapping techniques.


    1. Insert the MLCT cantilever into the Bruker Fluid Cantilever Holder, using tweezers and a preparation plate (Figure 1E).

    2. Make sure to slide the Cantilever back, until it takes up the entire space of the white base plate, and is properly held by the clamp (Figure 1F).

    3. Attach the Fluid Cantilever Holder to the BioScope Catalyst Scan Head, and adjust the laser spot to Cantilever B (rectangular), using the BioScope Catalyst EasyAlign table.

      Attention: Soft cantilevers are prone to strong thermal drift. Allow the cantilever to equilibrate, which can take up to 10 min. Do not continue until the laser spot has come to rest.

    4. In NanoScope software version 9.1, choose PeakForce QNM in Fluid (Standard Amplitude), from the Mechanical Properties Experimental Group.

    5. Determine the cantilevers spring constant, using the thermal tune technique, integrated in the NanoScope 9.1 software (Ohler, 2007).

    6. Bring the BioScope Catalyst Scan Head to a vertical position on the BioScope Catalyst EasyAlign table.

    7. Pipette a drop of approximately 30 µL of AIM V medium (without FBS) onto the cantilever holder. Make sure that the cantilever is fully submerged in the fluid, but is not damaged by the pipette tip during application of the medium.

    8. Bring the BioScope Catalyst Scan Head to a horizontal position, and allow the cantilever to equilibrate, which can take another 10–15 min. Then, readjust the laser spot.

    9. Mount an ethanol-cleaned glass slide on the measurement stage, and pipette a 50 µL drop of AIM V medium (without FBS) onto the center of the glass slide.

    10. Transfer the BioScope Catalyst Scan Head to the measurement stage, and engage the cantilever to the functionalized surface (a manual pre-approach can be done, until the drop of media on the cantilever holder and the media on the glass slide merge).

    11. Calculate the Deflection Sensitivity in V/nm, using the Update Sensitivity tool, which is integrated in the NanoScope 9.1 software.

    12. The system is calibrated and ready to measure.



    Figure 1. Experimental setup for CD4+ T cell sample and AFM preparation.

    A. Incubation of CD4+ T cells on functionalized surfaces (glass coverslips: top; PDMS substrate on glass slide: bottom). The samples were kept in a wet chamber during a 15-min incubation step at 37°C, to avoid drying (for more details on sample preparation, refer to Procedure part B, C, D and Video 1). B. Microscopic view of CD4+ T cells forming an IS on the functionalized substrate after 15 min of incubation. C. Functionalized glass coverslip mounted onto a glass slide after sample preparation. D. Functionalized piece of PDMS after sample preparation. E. Equipment used to insert a cantilever into the cantilever holder (I: tweezers, II: cantilever holder, III: preparation plate, VI: box of cantilevers). F. Detailed view of cantilever holder mounted onto the preparation plate, with adequately inserted cantilever.


  6. AFM based elasticity mapping

    1. Move the prepared sample (T cells attached to functionalized surfaces) to the measurement stage, and lock it in position with the magnetic Teflon clip.

    2. Mount the calibrated BioScope Catalyst Scan Head onto the measurement stage, and engage.

    3. Use an empty spot on the functionalized surface between the cells to set all parameters for elasticity mapping. In our experiments, deactivating the ScanAsyst Auto Control, and using the following parameters, showed the best results (if not mentioned here, the NanoScope default value was kept, or automatically set by the system):

      1. Starting scan size: 10 µm

      2. Line scan rate: 0.25 Hz

      3. Lines: 256

      4. Feedback gain factor: 0.5

      5. Peak Force Setpoint: 700 pN

      6. Peak Force Amplitude: 100 nm

    4. Several attempts are expected to establish contact between cantilever and surface. If this is difficult to achieve, increase the systems Engage Gain from factor 0.5 to factor 1.5, then withdraw and re-engage.

    5. While scanning, use the optical microscope (20× objective, leading to a 200-fold total magnification) to locate a T cell in close proximity, and approach it by shifting the cantilevers’ X-Y offsets, until approximately a quarter of the cell is covered by the cantilever movement (Figure 2A; see also Notes, for information regarding scanning whole cells).

      Attention: The manufacturer gives a relatively wide range of possible tip heights for MLCT cantilevers (2.5–8 µm). Therefore, collisions between the T cell and the front cantilever edges, leading to measurement artifacts, cannot be excluded. In our experiments, mostly the lower right or lower left cell quarter was approached, to make use of the cantilever tilt. However, some cells still turned out to be too large to scan, and some cantilevers turned out to have a tip too short to be used for this application (see also Notes 1, 2, Figure 2E).

    6. Use the NanoScope 9.1 “Capture next” or “Capture now” tools to save the data map (Figure 2B, C).

    7. If the cantilever is withdrawn from the surface before navigating to the next cell, make sure to set the cantilevers’ X-Y offsets to 0 again, to allow a maximum degree of navigational space within the X-Y movement range of 100 µm for the scan heads.


For a detailed introduction of PeakForce QNM data acquisition, we recommend the manufacturer’s video tutorial: https://www.youtube.com/watch?v=3I1Hl1gl3Uk.

Data analysis

  1. Extraction of elasticity data from elasticity maps (see Video 2 “Data Analysis”)

    1. Open the NanoScope 9.1 data map in the NanoScope Analysis 1.8 software, and switch to the Derjaguin-Muller-Toporov (DMT) modulus channel (Figure 2C), which represents elasticity (Derjaguin et al., 1975).

    2. Switch to the NanoScope Analysis “Bearing Analysis” tool, to mark square shaped spots on the elasticity map, covering the cellular structures of interest (see Note 3).

    3. Extract elasticity data from the Depth Histogram view, as XZ data in .txt format.

    4. Import the .txt data to a spreadsheet analysis software (e.g., Microsoft Excel), to calculate mean values and standard deviations of elasticity values.

    5. Wilcoxon matched-pairs signed rank tests can be used to test for statistical significance (e.g., using GraphPad Prism 6).


    Video 2. Data analysis and image export.


  2. Extraction of graphical images from elasticity maps (see Video 2)

    1. If necessary, crop the data map to the size that needs to be displayed.

    2. To extract images from the DMT modulus channel, right-click the scale and color axis next to the image, and select the “Choose Color” bar to choose a color scheme. To change the data scale, select the “Modify Scale” bar, and set the minimum and maximum values of the data scale that need to be displayed (e.g., 0–1,000 kPa).

    3. Use the “Export” Tool to save images from NanoScope Analysis software.

    4. To create image overlays in NanoScope Analysis (e.g., height profile overlayed with elasticity map), change to the height channel and to “3D Image”. Choose “Skin Type” from the image input menu, and select the DMT modulus channel (Figure 2D).

    5. Choose the preferred 3D image settings (e.g., zoom, rotation angle, label type), and export the image.

      For an overview of the complete experimental workflow and data analysis, including hands on and incubation times, see the Graphic abstract.



    Figure 2. AFM-based elasticity mapping (PeakForce QNM) and data analysis.

    A. Ideal cantilever orientation during elasticity mapping of a quarter CD4+ T cell, after shifting the X- and Y-offsets. The resulting elasticity map is indicated as a blue square, and the height profile as a black frame. B, C. Height profile and according elasticity channel (DMT) of a quarter of a T cell after data capture. Shown are scan size and elasticity data scale. D. 3D overlay of height profile and elasticity map, with scan dimensions and elasticity data scale. E. Examples of data maps showing measurement artifacts; top, left: lamellipodia region appears covered by blurry structures (single filopodia are unintentionally detached by the movement of the cantilever tip); top, right and bottom: blurry areas surrounding cell bodies (cantilever edge colliding with the cell body) (see also Notes 1, 2).

Notes

  1. When initiating the study, we were not aware of problems related to insufficient tip height and collision between the cell and the cantilever edges. Since we started with the cantilever type described, we kept that type during our measurements to guarantee comparability. Users that are initiating a new series of measurements might want to consider trying an alternative cantilever type. We suggest qp-BioT or qp-BioAC (Nanosensors, Neuchatel, Switzerland).

  2. Scanning whole cells is possible by increasing the scan size to 20–30 µm, since the IS forming T cells possess sizes of approximately 9–15 µm. In our experiments, we decided to focus on cell quarters, to minimize measurement artifacts due to collision between cantilever and cell (see also Note 2), and insufficient contact between the cell and the cantilever tip. These problems might be less severe with alternative cantilever types or other AFM systems.

  3. We strongly recommend analyzing several individual elasticity squares of the cellular structure(s) that needs to be addressed. This protocol was adapted from our Short Report “T cell stiffness is enhanced upon formation of immunological synapse” (see Jung et al., 2021). One focus of this work was to distinguish between the local stiffness of lamellipodia and cell body of CD4+ T cells. The data analysis strategy is given here as one possible option: for lamellipodia, three individual square-shaped surface segments of 500 × 500 nm were analyzed per cell. If very slender filopodia structures with a lateral width of less than 500 nm were seen, the segment size analyzed was reduced to 250 × 250 nm. To determine the elastic moduli of the cell bodies, one 1.5 × 1.5 µm surface segment was investigated.

Recipes

  1. Erythrocyte lysis buffer

    155 mM NH4Cl

    10 mM KHCO3

    0.1 mM EDTA

    in ddH2O, pH 7.3

Acknowledgments

We thank the Institute for Clinical Hemostaseology and Transfusion Medicine for providing donor blood; Carmen Hässig, Cora Hoxha, Gertrud Schäfer, Sandra Janku, and Mengnan Li for excellent technical help. This project was funded by the Deutsche Forschungsgemeinschaft (SFB 1027 projects A2 to BQ, B2 to MB, A12 to SI, and SPP1782 ID79/2-2 to SI), INM Fellow (to BQ).

The protocol presented in this manuscript was adapted from our previous work “T cell stiffness is enhanced upon formation of immunological synapse”, published in eLife 2021;10: e66643 (see Jung et al., 2021).

Competing interests

There are no conflicts of interest or competing interests.

Ethics

Research carried out for this study with healthy donor material (leukocyte reduction system chambers from human blood donors) is authorized by the local ethic committee [declaration from 16.4.2015 (84/15; Prof. Dr. Rettig-Stürmer].

References

  1. Basu, R., Whitlock, B. M., Husson, J., Le Floc'h, A., Jin, W., Oyler-Yaniv, A., Dotiwala, F., Giannone, G., Hivroz, C., Biais, N., et al. (2016). Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing. Cell 165(1): 100-110.
  2. Bromley, S. K., Burack, W. R., Johnson, K. G., Somersalo, K., Sims, T. N., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M. and Dustin, M. L. (2001). The immunological synapse. Annu Rev Immunol 19: 375-396.
  3. Derjaguin, B., Muller, V. and Toporov, Y. (1975). Effect of contact deformations on the adhesion of particles. J Colloid Interface Sci 53(2): 314-326.
  4. Desprat, N., Richert, A., Simeon, J. and Asnacios, A. (2005). Creep function of a single living cell. Biophys J 88(3): 2224-2233.
  5. Esensten, J. H., Helou, Y. A., Chopra, G., Weiss, A. and Bluestone, J. A. (2016). CD28 Costimulation: From Mechanism to Therapy. Immunity 44(5): 973-988.
  6. Friedmann, K. S., Bozem, M. and Hoth, M. (2019). Calcium signal dynamics in T lymphocytes: Comparing in vivo and in vitro measurements. Semin Cell Dev Biol 94: 84-93.
  7. Harrison, D. L., Fang, Y. and Huang, J. (2019). T-Cell Mechanobiology: Force Sensation, Potentiation, and Translation. Front Phys 7: 45.
  8. Hochmuth, R. M. (2000). Micropipette aspiration of living cells. J Biomech 33(1): 15-22.
  9. Hu, K. H. and Butte, M. J. (2016). T cell activation requires force generation. J Cell Biol 213(5): 535-542.
  10. Hui, K. L., Balagopalan, L., Samelson, L. E. and Upadhyaya, A. (2015). Cytoskeletal forces during signaling activation in Jurkat T-cells. Mol Biol Cell 26(4): 685-695.
  11. Husson, J., Chemin, K., Bohineust, A., Hivroz, C. and Henry, N. (2011). Force generation upon T cell receptor engagement. PLoS One 6(5): e19680.
  12. Jansen, K. A., Donato, D. M., Balcioglu, H. E., Schmidt, T., Danen, E. H. and Koenderink, G. H. (2015). A guide to mechanobiology: Where biology and physics meet. Biochim Biophys Acta 1853(11 Pt B): 3043-3052.
  13. Jung, P., Zhou, X., Iden, S., Bischoff, M. and Qu, B. (2021). T cell stiffness is enhanced upon formation of immunological synapse. Elife 10: e66643.
  14. Killian, J. L., Ye, F. and Wang, M. D. (2018). Optical Tweezers: A Force to Be Reckoned With. Cell 175(6): 1445-1448.
  15. Kummerow, C., Schwarz, E. C., Bufe, B., Zufall, F., Hoth, M. and Qu, B. (2014). A simple, economic, time-resolved killing assay. Eur J Immunol 44(6): 1870-1872.
  16. Loskill, P., Pereira, P. M., Jung, P., Bischoff, M., Herrmann, M., Pinho, M. G. and Jacobs, K. (2014). Reduction of the peptidoglycan crosslinking causes a decrease in stiffness of the Staphylococcus aureus cell envelope. Biophys J 107(5): 1082-1089.
  17. Majedi, F. S., Hasani-Sadrabadi, M. M., Thauland, T. J., Li, S., Bouchard, L. S. and Butte, M. J. (2020). T-cell activation is modulated by the 3D mechanical microenvironment. Biomaterials 252: 120058.
  18. Ohler, B. (2007). Cantilever spring constant calibration using laser Doppler vibrometry. Rev Sci Instrum 78(6): 063701.
  19. Pi, J. and Cai, J. (2019). Cell Topography and Its Quantitative Imaging by AFM. Methods Mol Biol 1886: 99-113.
  20. Scheuring, S. and Dufrêne, Y. F. (2010). Atomic force microscopy: probing the spatial organization, interactions and elasticity of microbial cell envelopes at molecular resolution. Mol Microbiol 75(6): 1327-1336.
  21. Taniuchi, I. (2018). CD4 Helper and CD8 Cytotoxic T Cell Differentiation. Annu Rev Immunol 36: 579-601.
  22. Thewes, N., Loskill, P., Spengler, C., Hümbert, S., Bischoff, M. and Jacobs, K. (2015). A detailed guideline for the fabrication of single bacterial probes used for atomic force spectroscopy. Eur Phys J E Soft Matter 38(12): 140.
  23. Trebak, M. and Kinet, J. P. (2019). Calcium signalling in T cells. Nat Rev Immunol 19(3): 154-169.

简介

[摘要] CD4 + T 細胞是協調針對細胞內病原體的特異性免疫反應和早期抑制腫瘤發展的重要參與者。 T 細胞的激活是由 T 細胞受體 (TCR) 的參與觸發的。在這裡,CD3 和 CD28 分子是關鍵因素,它們(共同)刺激信號通路,這些信號通路對 CD4 + T 細胞的活化和增殖至關重要。 T 細胞活化誘導 T 細胞和靶細胞之間形成緊密的機械結合,即所謂的免疫突觸 (IS)。因此,機械細胞特性,包括剛度,在調節細胞功能方面發揮著重要作用。過去,人們採用了許多方法來研究免疫細胞的機械特性,包括微量吸管、基於微孔板的流變儀、基於流式細胞儀變形的技術或使用光鑷。然而,在 IS 的亞細胞水平上,T 淋巴細胞的硬度仍然很大程度上難以捉摸。
通過該協議,我們引入了一種基於原子力顯微鏡 (AFM) 的方法,以研究 T 細胞在功能化玻璃/聚二甲基矽氧烷 (PDMS) 表面上的局部細胞剛度,該方法模擬靶細胞的局部刺激,從而誘導 T 細胞形成 IS。通過應用峰值力納米力學映射 (QNM) 技術,可以同時確定細胞表面結構和局部剛度,分辨率約為 60 nm。該協議可以很容易地適應研究影響 IS 形成和 T 細胞活化的眾多因素的機械影響。

圖形概要:


實驗工作流程概述。
各個實驗步驟顯示在左側, 每個步驟的手動操作和孵育時間顯示在右側。


[背景] T細胞屬於適應性免疫系統,可分為CD4 + T細胞或CD8 + T細胞。 CD4 + T 細胞對於協調免疫反應至關重要,而 CD8 + T 細胞,也稱為細胞毒性 T 淋巴細胞,是消除腫瘤和病原體感染細胞的關鍵參與者(Taniuchi,2018 年) 。 T 細胞通過 T 細胞受體 (TCR) 與匹配抗原的結合而被激活。因此,TCR 複合物的一個關鍵成分 CD3 分子轉導信號以激活下游通路,導致 T 細胞和靶細胞之間形成緊密連接,稱為免疫突觸 (IS)。此外,觸發白介素(例如,IL-6)產生的共刺激因子 CD28 的參與啟動了可持續激活和增殖所必需的信號通路(Esensten等,2016) 。除了受體-配體鍵的複雜芭蕾舞之外,細胞骨架的基本重排發生在 IS 形成過程中,包括微管組織中心 (MTOC) 向 IS 重新定向,以及 F-肌動蛋白環結構的形成在 IS。在這兩者之間,粘附分子淋巴細胞功能相關抗原 1 (LFA-1) 將其配體結合到靶細胞上,以密封和穩定 IS (Bromley等人,2001) 。在 T 細胞激活後,Ca 2+離子的細胞內濃度通過 Ca 2+流入顯著提高(Friedmann等人,2019) 。 Ca 2+在 T 細胞中充當重要的第二信使,以調節其活化、增殖和效應器功能(Trebak 和 Kinet,2019) 。
包括剛度和機械力在內的機械特性在調節細胞功能(包括 T 細胞的功能)中起著重要作用(Jansen等人,2015) 。最近的一項研究表明,細胞毒性 T 淋巴細胞通過施加機械力來優化其殺傷功能(Basu等人,2016) 。 T 細胞在激活後能夠產生皮牛頓 (pN) 範圍內的機械力(推力和拉力) (Husson等人,2011;Hu 和 Butte,2016) 。這種力的產生需要細胞內鈣的持續升高,以及功能性 F-肌動蛋白網絡和磷酸肌醇 3-激酶 (PI3K) 信號傳導的完整性(Hui等人,2015;Basu等人,2016) 。其他工作表明,T 細胞的激活需要機械力( Hu 和 Butte,2016 年) ,並且 T 細胞會對環境僵硬提示作出反應(Majedi等人,2020 年) 。細胞剛度的變化可以調節力的產生(Harrison等人,2019) 。然而,在 IS 的亞細胞水平上,T 淋巴細胞的硬度仍然很大程度上難以捉摸。
在過去的 20 年中,許多不同的方法被用於解決真核細胞,尤其是免疫細胞的僵硬和硬化過程。早期的方法,例如微量移液管抽吸和基於微孔板的流變測定法,涉及復雜的技術結構,其中細胞被插入或合併並受到機械應力(Hochmuth,2000;Desprat等人,2005) 。流式細胞術相關方法基於流動減速期間的變形或磁場中由膜結合金屬珠觸發的扭曲等原理。這些方法僅限於恆定的流體流來操作。相比之下,能夠在模擬生理條件的環境中表徵細胞彈性特性的方法,如細胞單層和細胞-細胞相互作用,能夠為生物力學過程提供更深入的見解。光鑷使用聚焦的激光束在 pN 範圍內產生吸引力或排斥力,被認為是表徵和修改生物材料的有前途的工具。因此,該方法主要用於移動或分類細胞,但仍未廣泛應用於局部細胞特性的表徵(Killian et al. , 2018) 。
原子力顯微鏡 (AFM) 是另一種生物物理方法,可用於確定 pN 範圍內的細胞力。 AFM 應用機械探針,通常由矽或氮化矽製成,尖端有納米薄的尖端,沿著細胞表面移動,並與定義的掃描網格的每個點相互作用。因此,它可以檢查高度輪廓,或分別在納米和 pN 範圍內以空間和力分辨率測量機械性能(Loskill等人,2014 年;Thewes等人,2015 年) 。近年來,AFM 已發展成為細胞生物學研究粘附力和細胞特性(如變形和彈性)的最重要工具之一(Scheuring 和 Dufrêne,2010;Pi 和 Cai,2019) 。
最近,我們建立了一種基於峰值力定量納米力學映射的方法,用於在亞細胞水平上同時確定 IS 形成過程中活 T 細胞的表面輪廓和剛度,分辨率約為 60 nm (Jung等人,2021) 。在這種方法中,針對 CD3、CD28 和 LFA-1 的抗體被固定在基質上,模擬靶細胞的局部刺激,從而在 T 細胞中誘導 IS 形成。該協議可以很容易地適應研究影響 IS 形成和 T 細胞活化的廣泛細胞內或細胞外因素的機械影響。

关键字:CD4+ T细胞, AFM, 僵硬度, 弹性图谱, 峰值力QNM



材料和試劑


1. 細胞培養瓶25 mL(Sarstedt,目錄號:83.3910.502)
2. 載玻片(Carl Roth,目錄號:H872.1)
3. 玻璃蓋玻片25 mm( VWR,目錄號:631-1584 )
4. Falcon ® 24 孔板( Corning,目錄號:353047 )
手術刀刀片(B. Braun Melsungen,目錄號:5518075)
6. Fluorodish Petri 培養皿 50 mm(World Precision Instruments,目錄號:FD35-100)
7. 聚丙烯管15 mL(Fisher Scientific,目錄號:11507411)
8. 聚丙烯管50 mL(Fisher Scientific,目錄號:10788561)
9. MLCT AFM 探針(Bruker AFM Probes,目錄號:MLCT-10)
10. 淋巴細胞分離培養基1077(PromoCell GmbH,目錄號:C-44010 )
11. 漢克斯平衡鹽溶液(Sigma-Aldrich,目錄號:H6648 )
12. CD4 + T細胞分離試劑盒人(Miltenyi,目錄號:130-096-533)
13. AIM V 細胞培養基(Thermo Fisher Scientific,Gibco,目錄號:31035025)
14. 胎牛血清(FBS)(Thermo Fisher,Gibco,目錄號:A4766801)
15. 磷酸鹽緩衝鹽水(PBS)(Thermo Fisher,Gibco,目錄號: 10010056)
16. 聚鳥氨酸(Sigma-Aldrich,Merck,目錄號: MFCD00286305 )
17. 抗LFA-1(ITGAL)抗體,小鼠單克隆(Antibodies-Online,目錄號:ABIN135680)
18. 小鼠抗人CD28抗體,小鼠單克隆(BD Pharmingen,目錄號:555725)
19. 小鼠抗人CD3抗體,小鼠單克隆(Diaclone,目錄號:854.010.000)
20. Sylgard 184 有機矽彈性體套件(Dow Europe GmbH,目錄號:1317318)
21. 對於替代懸臂(見註釋): qp-BioT/qp-BioAC(Nanoandmore GmbH,目錄號:qp-BioT-20/qp-BioAC-20)


設備


1. CO 2培養箱(Heracell Vios 160i, Thermo Fisher Scientific,目錄號: 51030403)
2. 離心機(Hettich Universal 32R,配備 50 mL 固定角轉子)
3. 離心機(Eppendorf,型號:5424,帶 2 mL 定角轉子)
4. AutoMACS Pro Separator(Miltenyi Biotec,目錄號:130-092-545)
細胞和粒子計數器(Beckman Coulter Z2,目錄號: 8043-30-0016 )
6. 靜電去離子器( Eltex Elektrostatik GmbH,目錄號: W0150L025U99)
7. 60°C PDMS 固化爐(Memmert,型號:100-800)
8. 隔膜實驗室泵(KNF,目錄號:N811KN.18)
9. 原子力顯微鏡(Bruker Corp.,型號:BioScope Catalyst)
10. 用於激光對準的布魯克校準表(布魯克公司,型號:Easy Align)
11. 熒光顯微鏡(Leica Microsystems,型號:DMI 4000 B)
12. 鑷子(例如,Agar Scientific,目錄號:AG5596-TI)
13. 懸臂支架(用於流體測量)(Bruker Corp.,型號:CAT-PCH)


軟件 


1. Research NanoScope,AFM 操作軟件(Bruker Corp.,版本 9.1,119071)
2. NanoScope 分析軟件(Bruker Corp.,1.8 版,132257)
3. Excel 2016(微軟)
4. GraphPad 棱鏡 6 (GraphPad)


程序


A. CD4 + T細胞的分離和培養
1. 如前所述(Kummerow et al. , 2014),從健康供體中製備外周血單個核細胞 (PBMC) 。簡而言之,為了從人血中分離單核細胞,我們使用淋巴細胞分離培養基,按照製造商的說明,在 450 g(加速:1,減速:0)和室溫(RT)下進行密度梯度離心 30分鐘。收集 PBMC,剩餘的紅細胞用1-3 mL 紅細胞裂解緩衝液(見配方)裂解1-2分鐘。預計持續時間:1.5 小時。
2. 根據製造商的協議,使用 AutoMACS Pro 分離器和人類 CD4 + T 細胞分離試劑盒從 PBMC 中負分離人類 CD4 + T 細胞。預計持續時間:1 小時。
3. 將分離的 CD4 + T 細胞以 3 × 10 6 /mL的密度重懸在具有 10% 胎牛血清 (FBS) (1 mL/孔) 的 AIM V 培養基的 24 孔板中,並將板保持在 37°含 5% CO 2的 C 24 小時。
4. 在 AFM 實驗之前,使用自動細胞計數器測量細胞密度,通過離心去除培養基(室溫下 1,200 rpm 5 分鐘)並將細胞懸浮液的密度調整為 2 × 10 5 – 3 × 10 5 /mL,通過在沒有 FBS 的 AIM V 培養基中重懸細胞。


B. 抗體功能化蓋玻片的製備(見視頻 1)
1. 清潔玻璃蓋玻片,用 70% 乙醇擦拭。
2. 使用永久性標記,在玻璃蓋玻片的背面標記一個直徑約為 7 mm 的中心點。
3. 用 40 μL 的聚鳥氨酸溶液(T 細胞的優化濃度:100 μg/mL,在滅菌的ddH 2 O中)在中心點塗上 30 分鐘。
4. 用隔膜實驗室泵取出聚鳥氨酸溶液,讓蓋玻片風乾 10 分鐘。
應用 20 µL 抗體稀釋液(在 PBS 中)並在 37°C 下孵育 30 分鐘,以使玻璃蓋玻片的一個點功能化。在 4°C 下儲存過夜。在我們的實驗中,以下抗體濃度組合顯示了 IS 形成的最佳結果(如果研究其他因素,最佳濃度可能會有所不同):
αLFA-1 (9 µg/mL)
αCD-3 (30 µg/mL)
αCD28 (90 µg/mL)


 
視頻 1. 樣品製備。


C. PDMS 的製備和 PDMS 表面的抗體功能化
1. 例如,培養皿、抹刀)緩慢地通過靜電去離子器的拱形(約 2 秒),以在使它們與聚二甲基矽氧烷 (PDMS) 彈性體組分 A(甲基氫矽氧烷-二甲基矽氧烷)接觸之前去除任何靜電荷,以及B(乙烯基封端的聚甲基矽氧烷)。
2. 在聚丙烯管中以 10:1 的比例混合組分 A 和 B,以創建剛度約為 2.5 MPa 的 PDMS(根據製造商的協議進行調整,如果解決了其他剛度問題)
注意:處理單個 PDMS 組件時,請嚴格使用聚丙烯材料(例如,VWR 刻度聚丙烯燒杯 100 mL,目錄號 213-3918;Sarstedt 攪拌棒 120 mm,PP,目錄號 81.970)和 Kimtech G3 純腈手套(例如, VWR 目錄號 112-4879)。
3. 將混合物倒入 50 毫米平底培養皿(例如Fluordish)中,並確保在表面覆蓋一層約 1.5 毫米厚。靜置 1 小時。
4. 放入培養箱中,在 60°C 下固化 16 小時。
在 AFM 測量之前,用手術刀刀片從 PDMS 層切割 10 × 10 mm 正方形,將 PDMS 片翻過來,然後繼續底部。
6. 繼續像玻璃蓋玻片 (B) 所述的功能化過程,並如下所述 (D) 應用單元格。


D. T 細胞在抗體功能化表面上的應用(見視頻 1)
1. 100 μL CD4 + T 細胞懸浮液移液到功能化表面的標記中心點(圖 1A,大約 2 × 10 4 – 3 × 10 4將被轉移)。
2. 讓細胞在濕室中沉降並與功能化表面相互作用 在 37°C 和 5% CO 2下放置 15 分鐘(塑料培養皿內襯濕紙巾;圖 1A - 1B),以避免干燥。
3. 將功能化玻璃蓋玻片安裝到載玻片上。在玻璃層之間移液器 2 μL 的滅菌 ddH 2 O,以確保功能化的蓋玻片或 PDMS 片通過凝聚力保持,並且在測量期間不會移動(圖 1C - 1D)。
4. 樣品應立即使用。丟棄樣品並在測量 60 分鐘後重新製備一個新樣品,因為細胞會死亡並在 RT 時開始從表面分離,並且不會增加部分 CO 2壓力。


E. AFM 準備和校準
注意:在我們的實驗中,NanoScope 9.1 軟件用於操作安裝在 Leica DMI 4000 B 光學顯微鏡上的 BioScope Catalyst 系統(Bruker,Santa Barbara,USA)。所有方法都專門針對這些系統進行了描述,但這些程序可以適用於其他軟件版本和 AFM 系統,從而允許使用峰值力攻絲技術。


1. 將MLCT 懸臂插入布魯克流體懸臂支架(圖 1E)。
2. 確保將懸臂向後滑動,直到它佔據了白色底板的整個空間,並被夾子正確固定(圖 1F)。
3. 將流體懸臂支架連接到 BioScope Catalyst 掃描頭,並使用 BioScope Catalyst EasyAlign 工作台將激光點調整到懸臂 B(矩形)。
注意:軟懸臂容易產生強烈的熱漂移。讓懸臂平衡,這可能需要長達 10 分鐘。在激光點停止之前不要繼續。
4. 在 NanoScope 軟件版本 9.1 中,從機械性能實驗組中選擇 PeakForce QNM in Fluid (Standard Amplitude)。
使用集成在 NanoScope 9.1 軟件(Ohler, 2007)中的熱調諧技術確定懸臂彈簧常數。
6. 將 BioScope Catalyst 掃描頭置於 BioScope Catalyst EasyAlign 工作台上的垂直位置。
7. 將大約 30 μL 的 AIM V 培養基(不含 FBS)移液到懸臂支架上。確保懸臂完全浸沒在流體中,但在應用介質期間不會被移液器尖端損壞。
8. 將 BioScope Catalyst 掃描頭置於水平位置,讓懸臂平衡,這可能需要10-15 分鐘。然後,重新調整激光光斑。
9. 在測量台上安裝乙醇清潔的載玻片,並將 50 μL 的AIM V 介質(無 FBS)滴移到載玻片的中心。
10. 將 BioScope Catalyst 掃描頭轉移到測量台,並將懸臂接合到功能化表面(可以進行手動預接近,直到懸臂支架上的介質下降和載玻片上的介質合併)。
11. 使用集成在NanoScope 9.1 軟件中的更新靈敏度工具計算以 V/nm 為單位的偏轉靈敏度。
12. 系統已校準並準備好進行測量。


 
圖 1. CD4 + T 細胞樣品和 AFM 製備的實驗裝置。 
A. 在功能化表面上孵育 CD4 + T 細胞(玻璃蓋玻片:頂部;載玻片上的 PDMS 基板:底部)。在 37°C 孵育 15 分鐘的過程中,將樣品保存在濕室中,以避免干燥(有關樣品製備的更多詳細信息,請參閱程序部分 B、C、D 和視頻 1)。 B. CD4 + T 細胞在孵育 15 分鐘後在功能化基底上形成 IS 的顯微鏡視圖。 C. 樣品製備後安裝在載玻片上的功能化玻璃蓋玻片。 D. 樣品製備後的功能化 PDMS。 E. 用於將懸臂插入懸臂支架的設備(I:鑷子,II:懸臂支架,III:準備板,VI:懸臂盒)。 F. 安裝在準備板上的懸臂支架的詳細視圖,懸臂已充分插入。


F. 基於 AFM 的彈性映射
1. 將準備好的樣品(附著在功能化表面的 T 細胞)移至測量台,並用磁性鐵氟龍夾將其鎖定到位。
2. 將校准後的 BioScope Catalyst 掃描頭安裝到測量台上,然後接合。
3. 在單元格之間的功能化表面上使用一個空白點來設置彈性映射的所有參數。在我們的實驗中,停用 ScanAsyst Auto Control 並使用以下參數顯示了最佳結果(如果此處未提及,NanoScope 默認值被保留,或由系統自動設置):
起始掃描尺寸:10 µm
線掃描率:0.25 Hz
行數:256
反饋增益係數:0.5
峰值力設定點:700 pN
峰值力幅值:100 nm
4. 預計會有幾次嘗試在懸臂和表面之間建立接觸。如果這難以實現,請將系統參與增益從 0.5 倍增加到 1.5 倍,然後退出並重新參與。
掃描時,使用光學顯微鏡(20倍物鏡,總放大倍數為 200 倍)定位附近的 T 細胞,並通過移動懸臂的 XY 偏移量接近它,直到覆蓋大約四分之一的細胞通過懸臂運動(圖 2A;另請參閱註釋,了解有關掃描整個細胞的信息)。
注意:製造商為 MLCT 懸臂 (2.5–8 µm) 提供了相對較寬的可能尖端高度範圍。因此,不能排除 T 細胞和前懸臂邊緣之間的碰撞,從而導致測量偽影。在我們的實驗中,大部分是接近右下角或左下角單元格,以利用懸臂傾斜。然而,一些細胞仍然太大而無法掃描,並且一些懸臂的尖端太短而無法用於此應用(另請參見註釋 1、2、圖 2E)。
6. 使用 NanoScope 9.1“下一個捕獲”或“立即捕獲”工具保存數據映射(圖 2B,C)。
7. 如果懸臂在導航到下一個單元之前從表面撤回,請確保再次將懸臂的 XY 偏移設置為 0,以允許掃描頭在 100 μm 的 XY 移動範圍內的最大導航空間度。


PeakForce QNM數據採集的詳細介紹,推薦廠商的視頻教程: https://www.youtube.com/watch?v=3I1Hl1gl3Uk 。




數據分析


A. 從彈性圖中提取彈性數據(參見視頻 2“數據分析”)
1. 在 NanoScope Analysis 1.8 軟件中打開 NanoScope 9.1 數據圖,並切換到代表彈性的 Derjaguin-Muller-Toporov (DMT) 模量通道 (圖 2C) (Derjaguin et al. , 1975) 。
2. 切換到 NanoScope 分析“軸承分析”工具,在彈性圖上標記方形點,覆蓋感興趣的細胞結構(見註 3)。
3. 從深度直方圖視圖中提取彈性數據,作為 .txt 格式的 XZ 數據。
4. 將 .txt 數據導入電子表格分析軟件(例如Microsoft Excel),以計算彈性值的平均值和標準差。
Wilcoxon 配對符號秩檢驗可用於檢驗統計顯著性(例如,使用 GraphPad Prism 6)。


 
視頻2. 數據分析和圖像導出。


B. 從彈性圖中提取圖形圖像(見視頻 2)
1. 如有必要,將數據圖裁剪為需要顯示的大小。
2. 要從 DMT 模數通道中提取圖像,請右鍵單擊圖像旁邊的比例和顏色軸,然後選擇“選擇顏色”欄以選擇顏色方案。要更改數據比例,請選擇“修改比例”欄,並設置需要顯示的數據比例的最小值和最大值(例如,0 – 1,000 kPa)。
3. 使用“導出”工具從NanoScope 分析軟件保存圖像。
4. 要在 NanoScope 分析中創建圖像疊加(例如,用彈性圖疊加的高度輪廓),請切換到高度通道和“3D 圖像”。從圖像輸入菜單中選擇“皮膚類型”,然後選擇 DMT 模數通道(圖 2D)。
選擇首選的 3D 圖像設置(例如,縮放、旋轉角度、標籤類型),然後導出圖像。
有關完整的實驗工作流程和數據分析的概述,包括動手和孵化時間,請參閱圖形摘要。
 
圖 2. 基於 AFM 的彈性映射 (PeakForce QNM) 和數據分析。
在移動 X 和 Y 偏移後,四分之一 CD4 + T 細胞的彈性映射期間的理想懸臂方向。生成的彈性圖用藍色方塊表示,高度輪廓用黑色框表示。 B、C. 數據採集後四分之一 T 細胞的高度剖面和相應的彈性通道 (DMT)。顯示的是掃描大小和彈性數據比例。 D. 高度剖面和彈性圖的 3D 疊加,具有掃描尺寸和彈性數據比例。 E. 顯示測量偽影的數據圖示例;左上圖:片狀偽足區域似乎被模糊結構覆蓋(單個絲狀偽足因懸臂尖端的移動而無意分離);頂部、右側和底部:細胞體周圍的模糊區域(懸臂邊緣與細胞體碰撞)(另見註釋 1、2)。


筆記


1. 在開始研究時,我們沒有意識到與尖端高度不足以及電池與懸臂邊緣之間的碰撞有關的問題。由於我們從所描述的懸臂類型開始,我們在測量期間保留了該類型以保證可比性。正在啟動一系列新測量的用戶可能想要考慮嘗試另一種懸臂類型。我們建議使用 qp-BioT 或 qp-BioAC(Nanosensors,Neuchatel,Switzerland)。
2. 通過將掃描尺寸增加到 20-30 µm 可以掃描整個細胞,因為形成 T 細胞的 IS 具有大約 9-15 µm 的尺寸。在我們的實驗中,我們決定專注於細胞四分之一,以最大限度地減少由於懸臂和細胞之間的碰撞而導致的測量偽影(另見註 2),以及細胞和懸臂尖端之間的接觸不足。使用替代懸臂梁類型或其他 AFM 系統時,這些問題可能不那麼嚴重。
3. 我們強烈建議分析需要解決的細胞結構的幾個單獨的彈性正方形。該協議改編自我們的簡短報告“免疫突觸形成後 T 細胞剛度增強”(參見Jung等人,2021 年)。這項工作的一個重點是區分片狀偽足的局部僵硬和 CD4 + T 細胞的細胞體。此處給出的數據分析策略是一種可能的選擇:對於片狀偽足,每個細胞分析三個 500 × 500 nm 的單獨方形表面片段。如果看到橫向寬度小於 500 nm 的非常細長的絲狀偽足結構,則分析的片段大小減少到 250 × 250 nm。為了確定細胞體的彈性模量,研究了一個 1.5 × 1.5 µm 的表面段。


食譜_


1. 紅細胞裂解緩衝液
155 毫米 NH 4氯
10 毫米碳酸氫鉀3
0.1 毫米乙二胺四乙酸
在 ddH 2 O, pH 7.3


致謝


我們感謝臨床止血和輸血醫學研究所提供獻血; Carmen Hässig、Cora Hoxha、Gertrud Schäfer、Sandra Janku 和 Mengnan Li 提供了出色的技術幫助。該項目由 Deutsche Forschungsgemeinschaft(SFB 1027 項目 A2 至 BQ、B2 至 MB、A12 至 SI 和 SPP1782 ID79/2-2 至 SI)、INM Fellow(至 BQ)資助。
本手稿中提出的方案改編自我們之前的工作“免疫突觸形成後 T 細胞剛度增強”,發表於 eLife 2021;10:e66643(見Jung等人,2021 年)。


利益爭奪


不存在利益衝突或競爭利益。
倫理


本研究使用健康供體材料(來自人類獻血者的白細胞減少系統室)進行的研究已獲得當地倫理委員會的授權 [2015 年 4 月 16 日的聲明(84/15;Rettig-Stürmer 博士教授)。


參考


1. Basu,R.,Whitlock,BM,Husson,J.,Le Floc'h,A.,Jin,W.,Oyler-Yaniv,A.,Dotiwala,F.,Giannone,G.,Hivroz,C.,Biais , N.,等人。 (2016 年)。細胞毒性 T 細胞使用機械力來增強靶細胞殺傷力。 165(1)號電池:100-110。
2. Bromley, SK, Burack, WR, Johnson, KG, Somersalo, K., Sims, TN, Sumen, C., Davis, MM, Shaw, AS, Allen, PM 和達斯汀, ML (2001)。免疫突觸。 Annu Rev Immunol 19:375-396。
3. Derjaguin, B.、Muller, V. 和 Toporov, Y. (1975)。接觸變形對顆粒粘附的影響。 J膠體界面科學53(2):314-326。
4. Desprat, N.、Richert, A.、Simeon, J. 和 Asnacios, A. (2005)。單個活細胞的蠕變功能。 生物物理學雜誌88(3):2224-2233。
Esensten, JH, Helou, YA, Chopra, G., Weiss, A. 和 Bluestone, JA (2016)。 CD28 共刺激:從機製到治療。 豁免44(5):973-988。
6. Friedmann, KS, Bozem, M. 和 Hoth, M. (2019)。 T 淋巴細胞中的鈣信號動力學:比較體內和體外測量。 精細胞開發生物學94:84-93。
7. Harrison, DL, Fang, Y. 和 Huang, J. (2019)。 T 細胞力學生物學:力覺、增強和翻譯。 前物理層 7:45 。
8. 霍克穆斯,RM(2000 年)。活細胞的微量吸管。 J Biomech 33(1):15-22。
9. Hu, KH 和 Butte, MJ (2016)。 T 細胞激活需要力的產生。 細胞生物學雜誌213(5):535-542。
10. Hui, KL、Balagopalan, L.、Samelson, LE 和 Upadhyaya, A. (2015)。 Jurkat T 細胞信號激活過程中的細胞骨架力。 分子生物學細胞26(4):685-695。
11. Husson, J.、Chemin, K.、Bohineust, A.、Hivroz, C. 和 Henry, N. (2011)。在 T 細胞受體接合時產生力。 公共科學圖書館一號6(5):e19680。
12. Jansen, KA, Donato, DM, Balcioglu, HE, Schmidt, T., Danen, EH 和 Koenderink, GH (2015)。機械生物學指南:生物學和物理學相遇的地方。 Biochim Biophys Acta 1853(11 Pt B):3043-3052。
13. Jung, P.、Zhou, X.、Iden, S.、Bischoff, M. 和 Qu, B. (2021)。 T 細胞硬度在免疫突觸形成後增強。 生命10: e66643 。
14. Killian, JL, Ye, F. 和 Wang, MD (2018)。光鑷:不可忽視的力量。 單元格175(6):1445-1448。
15. Kummerow, C.、Schwarz, EC、Bufe, B.、Zufall, F.、Hoth, M. 和 Qu, B. (2014)。一種簡單、經濟、時間分辨的殺滅試驗。 Eur J Immunol 44(6):1870-1872。
16. Loskill, P.、Pereira, PM、Jung, P.、Bischoff, M.、Herrmann, M.、Pinho, MG 和 Jacobs, K. (2014)。肽聚醣交聯的減少導致葡萄球菌的硬度降低 金黃色葡萄球菌細胞包膜。 生物物理學雜誌 107(5):1082-1089。
17. Majedi, FS, Hasani-Sadrabadi, MM, Thauland, TJ, Li, S., Bouchard, LS 和 Butte, MJ (2020)。 T 細胞活化由 3D 機械微環境調節。 生物材料252:120058。
18. Ohler, B. (2007)。使用激光多普勒測振法校準懸臂彈簧常數。 Rev Sci 儀器78(6):063701。
19. Pi, J. 和 Cai, J. (2019)。細胞形貌及其 AFM 定量成像。 方法 Mol Biol 1886:99-113。
20. Scheuring, S. 和 Dufrêne, YF (2010)。原子力顯微鏡:以分子分辨率探測微生物細胞包膜的空間組織、相互作用和彈性。 摩爾微生物75(6):1327-1336。
21. 谷內一(2018)。 CD4 輔助和 CD8 細胞毒性 T 細胞分化。 Annu Rev Immunol 36:579-601。
22. Thewes, N.、Loskill, P.、Spengler, C.、Hümbert, S.、Bischoff, M. 和 Jacobs, K. (2015)。用於製造用於原子力光譜的單個細菌探針的詳細指南。 Eur Phys JE 軟物質38(12): 140。
23. Trebak, M. 和 Kinet, JP (2019)。 T細胞中的鈣信號傳導。 Nat Rev Immunol 19(3):154-169。

  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Jung et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jung, P., Zhou, X., Iden, S., Qu, B. and Bischoff, M. (2022). Characterization of the Elasticity of CD4+ T Cells: An Approach Based on Peak Force Quantitative Nanomechanical Mapping. Bio-protocol 12(8): e4383. DOI: 10.21769/BioProtoc.4383.
  2. Jung, P., Zhou, X., Iden, S., Bischoff, M. and Qu, B. (2021). T cell stiffness is enhanced upon formation of immunological synapse. Elife 10: e66643.
提问与回复

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

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