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Dec 2018

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High Throughput Traction Force Microscopy for Multicellular Islands on Combinatorial Microarrays
组合微阵列多细胞岛的高通量牵引力显微镜实验   

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Abstract

The composition and mechanical properties of the cellular microenvironment along with the resulting distribution of cellular devolved forces can affect cellular function and behavior. Traction Force Microscopy (TFM) provides a method to measure the forces applied to a surface by adherent cells. Numerous TFM systems have been described in literature. Broadly, these involve culturing cells on a flexible substrate with embedded fluorescent markers which are imaged before and after relaxion of cell forces. From these images, a displacement field is calculated, and from the displacement field, a traction field. Here we describe a TFM system using polyacrylamide substrates and a microarray spotter to fabricate arrays of multicellular islands on various combinations of extra cellular matrix (ECM) proteins or other biomolecules. A microscope with an automated stage is used to image each of the cellular islands before and after lysing cells with a detergent. These images are analyzed in a semi-automated fashion using a series of MATLAB scripts which produce the displacement and traction fields, and summary data. By combining microarrays with a semi-automated implementation of TFM analysis, this protocol enables evaluation of the impact of substrate stiffness, matrix composition, and tissue geometry on cellular mechanical behavior in high throughput.

Keywords: Traction force microscopy (牵引力显微镜), TFM (TFM), Multicellular island (多细胞岛), Microarray (微阵列), Substrate stiffness (基底刚度), Microenvironment (微环境), Mechanobiology (机械生物学), Cellular forces (细胞力)

Background

Cellular generated forces are transmitted to the extracellular matrix (ECM) through integrins, and to surrounding cells by cadherins. The signaling associated with the transduction of these forces plays an important role in the regulation of proliferation, differentiation, morphogenesis, and homeostasis. Further, the magnitude and distribution of cellular forces are impacted by the conditions of the microenvironment such as ECM composition, geometry, substrate stiffness, and the presence of other ligands or drugs (Discher et al., 2009). We recently showed how ECM composition and substrate stiffness work together to affect liver progenitor cell differentiation and contractility (Kourouklis et al., 2016), and that cell contractility and traction stress distribution can facilitate spatial patterning of liver progenitor cell differentiation (Kaylan et al., 2018). Investigating the ways that the conditions of microenvironment such as ECM composition, geometry, substrate stiffness, and the presence of other ligands or drugs, mediate biomechanical and biochemical responses requires engineered systems that enable systematic control of the environment along with a way to assay the resulting behavior.

A variety of techniques have merged to measure cell-generated forces (Polacheck and Chen, 2016). Traction force microscopy (TFM) is a well-established method used to measure the forces applied by cells to the surface they are adhered to (Butler et al., 2002). While this concept has been expanded to three-dimensional cell culture, this protocol with only address TFM in two dimensional monolayers. Since its establishment, many authors have implemented TFM systems using various approaches and algorithms (Schwarz and Soine, 2015; Kulkarni et al., 2018). Broadly, in TFM, a flexible cell culture substrate is prepared with embedded fluorescent markers, often polymer beads. This substrate is modified such that cells will adhere, often by attaching a suitable ECM protein. Cells are then cultured on these substrates. Using an inverted fluorescence microscope, an image is taken the substrate surface, and the fluorescent markers, under each cell area of interest. The cells lysed using a detergent or enzyme, resulting in any forces applied to the surface by the cells to be relaxed. The same region is imaged once again. From these images, a displacement field is calculated using specialized software, based on the movement of the fluorescent markers after cell lysing. From the displacement field, a traction stress field is the calculated, using the known mechanical characteristics of the substrate.

In the MATLAB code provided with this protocol, the displacement field is calculated using a publicly available digital image correlation (DIC) algorithm (Landauer et al., 2018). Traction forces calculation is performed using an available Fourier transform traction cytometry (FFTC) algorithm (Sabass et al., 2008; Han et al., 2015). These methods were chosen based on their relatively low computational expense and time and relatively few required user inputs. This supports the high throughput nature of the analysis and goal to allow “off the shelf” use for labs who do not focus on TFM as a core competency. However, the code was intended to enable users to substitute alternative displacement or traction field algorithms based on the needs and computational resources of the user and application.

Performing TFM often involves bulk preparations of adhesive substrates which requires relatively large amounts of adhesive proteins, decreasing the practicality of performing TFM on multiple ECM proteins or surface bound ligands. Here we use contact printed microarrays which allows multiple ECM/ligand combinations, with replicates, to be included on a single substrate with low material usage (Flaim et al., 2005). TFM is often implemented either on single cells, or randomly distributed small colonies. In the microarray system, we can assess the mechanical behavior of multicellular islands where forces are transmit to the substrate as well as neighboring cells, resulting in collective behavior (Mertz et al., 2013). Further, these multi cellular islands are of consistent size and shape. This permits analysis of average mechanical behavior of these islands as a function of the environmental conditions. Island diameter can be tuned by using differently sized microarray pins, adding geometry as an additional parameter to investigate. The MATLAB code provided includes techniques to identify island boundaries and align data from replicate islands to enable analysis of these average behaviors. These cellular micro-arrays can be assayed in parallel using immunocytochemistry, which has been described in more detailed elsewhere (Kaylan et al., 2017), to allow correlation of mechanical and phenotypic behavior.

Our application has focused on the relationship between spatial patterns of mechanical behavior and differentiation of liver progenitor cells. This method can be applied to other stem cell systems especially where spatial patterning and collective mechanical behavior is of interest. It can also be applied to investigations of how the mechanical behavior of cancer relates to its microenvironment and further impacts proliferation or response to drugs. Although here we used contact microarray printing, this protocol can easily be applied to other micro patterned systems such as those with non-contact printing (Romanov et al., 2014) and Polydimethylsiloxane (PDMS) stamped substrates (Kane et al., 1999). In this application, we utilize the XY position within the array to know the condition of that island. This protocol could be extended to other systems where spatial position of cells on their substrate is linked to an experimental variable or substrates such as with gradients of stiffness (Hadden et al., 2017) or biomolecules (Dertinger et al., 2002).

Materials and Reagents

  1. Aluminum foil
  2. Forceps
  3. Pipettes
  4. 1 ml syringe
  5. 6-well polystyrene microplates ( Fisher Scientific, catalog number: 08-772-1B)
    Note: Other 6-well microplates may be suitable, however the 35 mm glass bottom Petri dishes fit well into these plates and do not fit into all 6-well plates.
  6. Far-red fluorescent beads, 0.2% v/v (Life Technologies, catalog number: F-8816)
  7. 12 mm glass coverslip (Electron Microscopy Sciences, catalog number: 72231-01)
  8. 384-well polypropylene plate (USA Scientific, catalog number: 1823-8400)
  9. 35 mm glass bottom dish with 13 mm well and #1.5 German cover glass (Cell E&G, catalog number: GBD00002-200)
  10. Ligands
    Fc-recombinant DLL1, mouse (R&D Systems, catalog number: 5026-DL-050 )
    Fc-recombinant DLL4, mouse (AdipoGen , catalog number: AG-40A-0145-C050)
    Fc-recombinant JAG1, rat (R&D Systems , catalog number: 599-JG-100)
    Human IgG (R&D Systems, catalog number: 1–001-A)
    Protein A/G (Life Technologies, catalog number: 21186)
    Note: Cell-cell signaling ligands can be arrayed on the surface in combination with ECM proteins. Retention of smaller molecules in the gel may differ, therefore it may be necessary to conjugate ligands to an additional molecule. We used Fc-recombinant notch ligands conjugated to protein A/G, and IgG as a control. If using this strategy, ligands should be combined with protein A/G at a molar ratio of 6:1 or greater. Ligands should be reconstituted per manufacturer recommendations and stored at -80 °C. 
  11. Extracellular Matrix proteins
    Collagen I, rat tail (EMD Millipore, catalog number: 08-115MI), stored at 4 °C
    Collagen III, human (EMD Millipore, catalog number: CC054)
    Collagen IV, human (EMD Millipore, catalog number: CC076)
    Fibronectin, human (Sigma-Aldrich, catalog number: F2006)
    Laminin, mouse (EMD Millipore, catalog number: CC095)
    Note: Numerous ECM proteins are suitable for this process. Choice of ECM protein will depend on the application. Here we have listed those used in our in various studies, which were prepared at 1 mg/ml, and stored at -20 °C unless otherwise noted. 
  12. 3-TPM (Sigma-Aldrich, catalog number: 440159-100ML)
    Note: Exposure of 3-TPM to air could negatively impact silanization of glass substrates and should be stored per manufacturer instructions. The 3-TPM described here is packaged in a Sure/SealTM container, which requires a syringe and needle for use.
  13. NaOH (Sigma-Aldrich, catalog number: 415413-1L)
  14. Ethanol 200 Proof (Decon Labs, catalog number: UN1170)
  15. Acrylamide (Sigma-Aldrich, catalog number: A3553-100G)
  16. Bis-acrylamide (Sigma-Aldrich, catalog number: M7279-25G)
  17. Irgacure 2959 (BASF Corporation, catalog number: 55047962)
  18. Methanol (Sigma-Aldrich, catalog number: 179957-1L)
  19. Sodium acetate (Sigma-Aldrich, catalog number: S2889-250G)
  20. Ethylenediaminetetraacetic (EDTA) (Sigma-Aldrich, catalog number: ED-100G)
  21. Glycerol (Sigma-Aldrich, catalog number: M6145-25ML)
  22. Glacial acetic acid (Sigma-Aldrich, catalog number: 695092-500ML)
  23. Phosphate-buffered saline (PBS) (Fisher Scientific, catalog number: SH3001302)
  24. 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) (Sigma-Aldrich, catalog number: C3023-1G)
  25. Dextran-rhodamine (Life Technologies, catalog number: D-1841)
  26. 100x penicillin-streptomycin solution ( Fisher Scientific , catalog number: SV30010)
  27. Bovine serum albumin (Sigma-Aldrich, catalog number: A2153-100G)
  28. Sodium dodecyl sulfate (Fisher Scientific, catalog number: BP166-100)
  29. Triton X-100 (Sigma-Aldrich, catalog number: X100-1L)
  30. 2x ECMP Printing Buffer (see Recipes)
  31. 2x Growth Factor (GF) Printing Buffer (see Recipes)
  32. Polyacrylamide Prepolymer Solution (see Recipes)

Equipment

  1. Vacuum Desiccator (Fisher Scientific, catalog number: 08-642-5)
  2. OmniGrid Micro arrayer (Digilab)
    Note: Other microarrayers with similar capabilities can be used instead. Follow manufacturer’s instructions to operate arrayer.
  3. Stealth or solid pins (ArrayIt, catalog number: SMP3 or SSP015)
    Note: Other pin types may be suitable for use ArrayIt, SMP3 prints spots approximately 150 μm in diameter. SSP015 pins print spots of approximately 600 μm in diameter. Select pins that produce an appropriate island size for your application.
  4. UV crosslinker, UVP, CL-1000
  5. Vortex
  6. Fluorescence microscope, Zeiss Axiovert 200M with a computer running Zeiss Zen image acquisition software
    Note: Here we use a Zeiss Axiovert 200M, however other fluorescent microscopes are suitable. The microscope should be equipped with a robotic stage and the ability to mark and find locations. We also advise environmental controls (i.e., 37 °C and 5% CO2 since TFM involves live cell imaging. The provided MATLAB code is designed to directly load and read metadata from the Zeiss proprietary format (.czi). However, because it utilizes tools from Bio-Formats (Linkert et al., 2010), files from most common microscopes can be loaded with little or no modifications.

Software

  1. MATLAB version 2018b, Mathworks 
  2. MATLAB scripts from https://github.com/UnderhillLab/High-Throughtput-TFM
    Note: The provided scripts are intended to be able to be used by those with basic MATLAB experience, however adjusting the scripts for the additional needs of a given user may require more advanced MATLAB expertise.

Procedure

  1. Substrate fabrication
    Polyacrylamide gels are prepared as adapted from protocols in literature (Tse and Engler, 2010; Wen et al., 2014).
    1. Silanization of glass bottom dishes. This functionalizes the glass surface so that the polyacrylamide hydrogel will covalently bind to it. 
      1. Add 250 μl of a 0.2 N NaOH solution to the glass area of each glass bottom dish. Ensure NaOH solution coats entire glass area but not the plastic surface as shown in Figure 1A.
      2. Incubate on a gently rocking shaker for 1 h.
      3. Rinse dishes 3 times with deionized water (dH2O). 
      4. Use compressed, filtered air to dry the dishes. Bake the dishes at 80 °C on aluminum foil on a hot plate until completely dry. Overheating the dishes may cause adhesive on the glass to fail. 
      5. Add 0.2 ml of 3-(Trimethoxysilyl) Propyl Methacrylate (3-TPM) to 9.8 ml of 100% ethanol.
      6. Apply 100 ul of 3-TPM solution to the glass area of each dish. Ensure 3-TPM solution coats entire glass area but not the plastic surface as shown in Figure 1A.
      7. Incubate on a gently rocking shaker for 30 min.
      8. Add 3 ml of ethanol to each dish. Incubate on a gently rocking shaker for 5 min. 
      9. Remove ethanol. Use compressed air to dry the dishes. 
      10. Bake the dishes at 80 °C on aluminum foil on a hot plate until completely dry.
        Note: At this stage, silanized glass bottom dishes can be stored for up to one month in a dark, dry location.


        Figure 1. Glass bottom dish silanization and gel fabrication. A. Proper coverage for NaOH and 3-TPM solutions. B. Droplet of prepolymer solution. C. Coverslip floated on prepolymer droplet. D. Inverted dish to allow beads to settle. E. Dish filled with deionized water following UV exposure. F. Dish with coverslip removed and gel dehydrated.

    2. Polyacrylamide gel fabrication. The polyacrylamide gel provides the deformable hydrogel substrate for cells to bind to as part of TFM preparation.
      1. Prepare a polyacrylamide pre-polymer solution based on desired stiffness (Recipe 3). You will need 20 μl of solution for each dish. 
      2. Add 0.1 g of Irgacure 2959 (light-sensitive) to 0.5 ml 100% methanol to produce a 20% w/v Irgacure solution. Vortex until solution is clear.
      3. Combine the pre-polymer solution and Irgacure solution at a 9:1 ratio. Prepare enough. 
      4. Sonicate the bead suspension for 15 min.
      5. Add beads to the working solution at a 1:500 ratio (i.e., 1 μl bead solution to 500 μl working solution). Vortex to ensure complete mixing. 
      6. Degas the full working solution in a vacuum desiccator for 15 min. 
      7. Pipette 20 μl of the working solution into the center a glass bottom dish. Avoid bubbles in the droplet as shown in Figure 1B.
      8. Carefully float a 12 mm coverslip onto the droplet. The solution should spread the edges of the droplet as shown in Figure 1C. 
      9. Repeat with the remaining dishes.
      10. Invert the dishes, as shown in Figure 1D, and let stand for 15 min to allow beads to migrate towards the surface.
      11. Expose dishes to 365 nM UVA for 15 min in the UV crosslinker. 
      12. Fill dishes with deionized water as shown in Figure 1E. Let sit for 3-24 h. 
      13. Carefully remove coverslips using forceps. Avoid twisting or shearing the coverslip which may cause ripping in the gel surface.
        Note: Use of clean coverslips and careful removal after 24 h of soaking in dH2O typically results in successful removal of the coverslip without damaging the gel. For very soft gels, it may be necessary to further passivate the coverslip surface. Strategies include wiping the surface with Rain‑X® using a delicate task wiper, or vapor deposition of Dichlorodimethylsilane.
      14. Dehydrate gels at 50 °C on a hot plate until all water has evaporated from the gel. A dehydrated gel is shown in Figure 1A. Gel dishes can be stored for up to one month in a dark, dry location. 

  2. Microarray printing
    This process prints an array of circular spots of biomolecules onto the polyacrylamide substrates. These array spots are where cells will adhere forming circular islands.
    1. If needed, prepare a suitable 2x printing buffer (Recipe 1)
      Note: ECM protein printing (ECMP) buffer is appropriate for most ECM molecules. Growth factor (GF) printing buffer is suitable for other classes of molecules such as growth factors or ligands, where a low pH could cause issues.
    2. Prepare a microarraying source plate.
      1. In a 384-well V-bottom microplate, prepare printing solutions which should include equal volumes of 2x printing buffer with biomolecule solution at double the target concentration. For many ECM proteins, 250 µg/m is a suitable concentration. Optimal concentrations will vary depending on the molecule, its retention, and its function. The total volume in each well should be between 5 and 15 µl. 
      2. In your source plate, you should also include a solution with a fluorescent marker which will be used to convey the orientation of the array to determine the locations of each condition. We recommend rhodamine-conjugated dextran at a final concentration of 2.5 mg/ml. The source plate configuration will differ based on the arrayer, pin configuration, and desired array layout.
      3. Mix each well thoroughly by pipetting. Take care to avoid generating bubbles. Centrifuge the source microplate for 1 min at 1,000 x g. Source plates can be used immediately or stored at 4 °C for 1 day before until microarray fabrication. If storing, cover source plate with an adhesive seal. 
      4. Clean and prepare pins according to the manufacturer's instructions. Load cleaned pins into the microarrayer printhead.
      5. Prepare the microarrayer and arraying program using the manufacturer’s software. The set-up and programming will differ based on the arrayer and desired array layout. The program should be devised such that the array orientation is unambiguous, and the locations of each arrayed conditions are known and could be determined by the location relative to the fluorescent marker in any orientation. An example of this is provided in Figure 2.
        Notes:
        1. The example array layout shown was designed for printing 6 replicates of 4 conditions, which were simultaneously printed by four SSP015 pins which produce 600 μm diameter islands. The number of islands and/or conditions can be scaled up when using pins that produce smaller islands.
        2. Be sure to record how the layout corresponds to the arrayed conditions. Include wash steps using both water and dimethyl sulfoxide (DMSO) between each condition in order to prevent carry-over and cross-contamination.


        Figure 2. Example layout of an array of 4 ECM conditions with dextran rhodamine markers. When the islands and dextran rhodamine spots are visible, the orientation of the array can be discerned whether rotated or if viewed from above or below.

      1. Turn on the humidifier and ensure it is adjusted to 65% RH (non-condensing). Wait until the rheometer matches the set point. 
      2. Place the source plate in the arrayer in an appropriate adaptor.
      3. Dehydrate hydrogel substrates at 50 °C for 15 min. Protect from light due to the presence of the fluorescent beads. 
      4. Place dishes into an appropriate adaptor. If the arrayer can fit a standard multiwell plate, the 6-well plate is suitable to hold the dishes.
        Note: See the note under Materials and Reagents #5. 
      5. Begin array fabrication. Check frequently that the humidity has not dropped below 65% RH (non-condensing). 
      6. When the program is complete, store fabricated arrays covered with aluminum foil at room temperature and 65% RH (non-condensing) overnight. While the array spots are visible, it is helpful to visibly mark the top or bottom of the array so the orientation is known when placing on the microscope. For some hydrogel and pin combinations, it may be necessary to store arrays at ambient temperature and humidity for an additional two days to ensure arrays have dried completely. Arrays can be stored for up to 7 days before use. 

  3. Seeding cells on microarrays
    Here cells are transferred from their normal culture condition on to the microarrayed hydrogel substrates for TFM.
    1. To sterilize the gels, add 3 ml of PBS with 1% v/v penicillin/streptomycin. Expose to UV C for 30 min. Exchange penicillin/streptomycin solution for cell culture media. 
    2. Collect and count cells following the cell appropriate procedure. Resuspend cells in culture media at an appropriate concentration for seeding. This will differ based on cell type but will likely range between 170 x 103 and 7 x 105 cells/ml. Add 3 ml of cell suspension to each dish. Incubate dishes at 37 °C and 5% CO2 for 2-24 h, or until confluent cell islands have formed. Seeding density and time may need to be optimized for your cells and application. Agitation of the dishes every 15-60 min may also aid in forming consistent, confluent islands.
    3. Once islands have formed, rinse arrays twice with 3 ml prewarmed media. At this stage, add any experimental treatments such as growth factors or inhibitors. Change media every 1-2 days until time to perform TFM, or as your cell culture protocol suggests, maintaining any treatment concentrations at each exchange. Figure 3 shows an example of an array with cellular islands.


      Figure 3. 35 mm glass bottom dish with cellular island array after 24 h of culture

  4. Acquiring images for traction force microscopy
    1. Prepare a solution of 1% v/v bovine serum albumin (BSA) and 1% v/v sodium dodecyl sulfate (SDS) in PBS. This will be used to dissociate cells during image acquisition. 
    2. The microscopy process will vary based on the microscope used, but the following steps should broadly apply. Some additional details are provided for use with Zeiss Zen software. The microscope should have a robotic stage and a chamber which provides incubation and humidity control which have been appropriately set and allowed to come to equilibrium. Place one dish on the stage of the inverted fluorescent microscope. It is helpful to use the mark provided after array fabrication to place the dish with the array’s XY axis approximately aligned along that of the microscope stage. Secure the dish in place so that it does not shift when the stage moves.
    3. Using the appropriate fluorescent channel, locate and mark (in the software) the XY positions of the fluorescent arrayed markers. In Zeiss Zen Blue 2, this is accomplished in the acquisition section by checking the tiles box and adding a single position for each island, as shown in Figure 4.


      Figure 4. Zeiss Zen Blue 2 environment. To mark locations, in acquisition, mark Tiles and enter advanced setup. Set individual island positions as shown by the yellow markers. The verify positions tool can be used to move through positions to set the Z position without altering the XY position. 

    4. Using phase contrast or bright field, locate and mark the XY positions of the cell islands. Either imaging modality may be suitable as long as the image can provide the boundary of the cell island in subsequent analysis. Individually find and save the Z plane focus of each island. An example image phase contrast is shown in Figure 5A. The verify positions tool can be used to move through positions to set the Z position without altering the XY position. The saved XY positions of the islands can be saved and reused for subsequent dishes.
    5. Begin the automated imaging of the phase contrast images of the cell islands. Save this file with a suitable name that notes the experiment details as well as that it is the phase contrast image.
    6. Switch to the appropriate fluorescent channel for the beads. Individually find and save the Z plane focus of the top surface of the gel under each island. Take care to avoid changing the XY positions. Save this file with a suitable name that notes the experiment details as well as that it is the pre-dissociation image. An example image is shown in Figure 5B.


      Figure 5. Example images from TFM data collection. A. Phase contract image of a cellular island. Image should be clear enough to define island boundaries. B. Fluorescence image of the beads at the surface of the substrate under the island. Scale bar: 200 μm.

    7. Begin the automated imaging in the fluorescent channel of the pre-dissociation bead images. Save this file with the same experiment details as well as that it is the pre-dissociation image.
    8. Carefully add 150 µl of the SDS solution to the dish, taking care to not bump or move the dish. Monitor dissociation of the cell islands using the phase contrast channel. Wait until the islands have completely dissociated from the substrate at which time there the island locations, when viewed in phase contrast channel, should appear mostly blank.
      Note: Some cells may require addition of more SDS solution, or higher concentration. Additionally, Triton-X may be used instead. 
    9. Return to the island positions and using the fluorescent channel check that the beads at the top of the surface are still in focus. If the beads have moved out-of-plane due to relaxion of the cell induced deformation, correct and save the Z-coordinate of the focus plane but do not alter the XY positions. Repeat automated imaging of the islands to capture the post-dissociation images of the beads. The displacement of the beads will likely be difficult to discern, meaning the post-dissociation fluorescent bead image should appear very similar to the pre-dissociation image. Save this file with a suitable name that notes the experiment details as well as that it is the post-dissociation image.
    10. Repeat Steps D2-D9 for each dish. 

Data analysis

  1. Process images to estimate displacement fields and calculate traction fields
    Note: Images collected per the previous section may be exported and used with available TFM code packages. Here we provide the TFM code we have implemented, and its use is explained in the following sections. The TFM and analysis are designed to be used with arrays of cells with the same geometry to enable assessing replicates of the same condition. These scripts allow the user to generate TFM data and create basic visualizations. Further manipulation and analysis of the data for a user’s specific application may require generation of additional scripts. 

    1. Ensure the provided MATLAB code has been saved to an appropriate location. Navigate to the folder where this directory has been saved. 
    2. Make sure all image files have been saved or transferred to a folder available from the computer to be used for analysis. 
    3. From the command window, run the function run_island_tfm with no inputs.
    4. You will be prompted to select the file with the phase contrast image. Navigate to and select the file with the phase contrast image.
    5. A new file selection window will open in the same directory as the selected file. Now select the file with the pre-dissociation fluorescent image. Finally, when prompted, select the post-dissociation image.
    6. A GUI will appear for entering the experimental information for the image files. This GUI is shown in Figure 6. The graph panel shows the XY position of each image collected. The numbers correspond to how the images are indexed in the image file. Enter all relevant experiment information listed above the “Data file name” field. The pixel size will be read from the metadata of the image but can be changed. The “number of arrayed conditions” refers to the number of different conditions in the imaged array. For the example layout in this protocol, there are 4 arrayed conditions. This number is used to group the islands into their conditions.


      Figure 6. GUI for entering experiment and dish information. Chart panel shows XY position of each island, labeled by the index of the island in the file. Chart title is the image file name. Pixel size is pulled from the image metadata but can be set manually. All other fields are set by the user.

    7. Once these fields are completed, click “Set Info.” This will populate the “Data file name” field using the entered information which will be the file name of the output file. This field can be changed manually. Two additional fields will appear. Enter a name for the first condition in the “Condition 1” field. Enter the numbers of all the islands assigned this condition in the “Islands with Condition 1” field as a list of numbers. Click “Set Condition.” If there are additional conditions, repeat these steps for each of the remaining conditions. Once all conditions have been set, the “Done” button will appear. Confirm that all experiment information is correct, then click “Done.” All islands assigned a condition in this step will be analyzed. To analyze only some islands, set the number of conditions to the number conditions represented by the islands to be analyzed, and only list those islands in the “Islands with Condition” field.
    8. The script will now cycle through the conditions and islands. It will first run a script to correct frame shifts between the before and after dissociation.
      Note: The correct shift function is to remove full frame shifts between the images caused stage drift between collecting the images. This is done by performing digital image correlation on regions of the image far enough from the cells where there is no expected deformation. The script automatically selects four regions near the four corners of the image. To manually select these regions, change the ‘n’ to a ‘y’ in the frame_shift function call.
      A GUI will now appear to aid in drawing a boundary around the cell island. This GUI is shown in Figure 7. The software will attempt to draw a boundary around the cell island, which is plotted in red. The slides can be used to adjust the parameters of the trace which will cause the trace to rerun in real time. You can also change the values of these parameters by entering numbers into the text boxes and then hit the “Rerun” button. Alternatively, you can draw the boundary manually by clicking the “draw manual” button. Left click around the island until the boundary is closed. Double click inside the boundary to create the shape. To avoid sharp corners, after the polygon is drawn, a blurring and rounding is applied using the current value in the “Blur” field. To avoid this rounding, set this field to 1. The manual draw function can be used to trace multiple areas. To reset the manual boundaries, click “Clear boundaries,” or click “Rerun” to repeat the automated tracing. When satisfied with the boundary, click “Done.”


      Figure 7. GUI for selecting cell island boundary. Parameters can be set with the sliders which will update the boundary in real time, or by typing in values and clicking Rerun. You can also manually draw the boundary.

    9. This will bring up the next island. Repeat this process for all islands in the file selected for analysis. 
    10. When the boundary trace has been completed for each island, the data will be saved. The program will then move to calculate the displacement and traction fields for each island. This process can take up to several hours depending on the number of images, image size, and processors on the computer. Data is saved after each island to limit data loss in the case of issues during analysis. Data is saved in the folder “data out.” 
    11. Most of the computation time is the displacement field estimation. To rerun the traction field calculation with different settings on a file which already has the boundaries identified and displacement field calculated, run the command run_island_tfm(‘rerun’). You will be prompted to choose a data file which will be reanalyzed. 

  2. View data and generate summaries
    1. In the code provided, the output is saved in the folder “data out” with the file name established during analysis. To explore data from a single file, data can be loaded into the workspace by double clicking in the Current Folder explored or using the load function.
    2. This loads the cell array “all_cell_data” into the workspace. Each cell corresponds to a single island from the analysis. The output is intended to provide easy access to all relevant data for the user to explore and analyze in MATLAB or export to other programs for analysis as appropriate for their application. Table 1 provides the organization of the data stored within the output file. Data structure elements can be accessed using dot notation of the form structName.fieldName.
      Note: See https://www.mathworks.com/help/matlab/matlab_prog/access-data-in-a-structure-array.html for more information on accessing data in structures.
    3. A set of functions are provided to view and analyze data. Example code to use these functions is provided in data_analysis_examples. To view the results from a single island, use view_one_island. See code for further documentation. An example of the output is shown in Figure 8.


      Figure 8. Example output from view_one_island. Island information is displayed. Vector fields are plotted over the magnitudes of the displacement filed (in μm) and traction field (in Pa). The island boundary is plotted in black. The phase contrast image is also displayed, with the boundary plotted in yellow. The best fit ellipse is shown in red, with the major and minor axes.

    4. Using the boundary traced of the island, the script finds a best fit ellipse. This ellipse can be used to align compare many replicates of islands with the same geometry.
      Note: The provided scripts assume the islands can be represented by an ellipse. This can be adapted to other geometries by altering how the center and angle of rotation are identified.
      Use view_one_island_rotandcen to view the data centered and rotated according to the best fit ellipse.
    5. The function collect_island_data is provided to collect data from multiple islands across multiple TFM runs. The output of this function is a table with data and information on each of the islands loaded from the files (see Table 1), and a structure holding the displacement and traction data, indexed according to the “summ_ind” field of the summary table This table can be exported to excel or similar. When the function is run without an input, you will be prompted to select data files to load and consolidate. 

      Table 1. Structure of data output file and description of saved data
      Data Field


      Description
      cell_info.
      young

      Youngs Modulus of substrate

      pois

      Poisson Ratio of substrate

      pixelsize

      Size of original image pixels in micron

      experiment

      Description of experiment

      date_ID

      Identifier of experiment or run

      soluble

      Soluble factors or treatments added to dish

      dish_num

      Identifier of dish, e.g., if replicates of a unique condition

      time

      Time from seeding and TFM data collection

      arrayed_condition

      ECM or other condition of the island within the array

      Xposition

      X position of island within array

      Yposition

      Y position of island within array

      image_number

      Index of image within the original file

      file

      file containing original phase/brightfield image

      dm

      Final spacing, in pixels, of displacement/traction field output




      Images.
      Images{1}

      Pre-dissociation fluorescent image

      Images{2}

      Post-dissociation fluorescent image

      Images{3}

      Phase/Brightfield image




      cell_boundaries.
      mask

      mask of regions determined within island boundary

      boundary_points
      Cell containing the XY coordinates of each of the boundaries traced

      cny

      Sensitivity factor used to trace boundary

      sigma

      Blur factor used to trace boundary

      di

      Erosion factor used to trace boundary

      ellipse_fit.
      phi           
      Angle of roation of the fit ellipse with respect to the major axis being horizontal


      X_center
      center at the X axis of the non-rotated ellipse

      Y_center center at the Y axis of the non-rotated ellipse
             X0
      center at the X axis of the rotated ellipse


      Y0
      center at the Y axis of the rotated ellipse


      major_axis
      major axis of ellipse (in pixels)


      minor_axis 
      minor axis of ellipse (in pixels)

      rotated_boundary_points         

      Cell containing the XY coordinates of each of the boundaries traced, rotated and centered according to the fit ellipse




      cell_displacements.        
      raw_displacements. raw_displacements{1}       X component of raw estimated displacement field in pixels


      raw_displacements{2}         
      Y component of raw estimated displacement field in pixels


      raw_displacements{3}    
      Magnitude of raw estimated displacements in pixels

      Displacements.
      displacements{1}
      X component of displacement field in micron


      displacements{2}
      Y component of displacement field in micron


      displacements{3}       
      Magnitudes of displacements field in micron

      rotated_displacements

      displacements which have been rotated and centered using the fit ellipse




      cell_tractions.
      Tractions.
      tractions{1}
      X component of tractions in Pa


      tractions{2}
      Y component of tractions in Pa


      tractions{3}
      Magnitudes of tractions in Pa

      RMS

      Root mean square of traction field

      RMS_int

      Root mean square of traction field within cell island boundary

      Strain_Energy

      Strain Energy

      rotated_tractions

      Tractions which have been rotated and centered using the fit ellipse

    6. The output of the collect_island_data function can further be used to create summary data. The function summarize_islands plots and outputs averaged displacement and traction fields, see Figure 9 for an example. This function uses the aligned the data from each island based on the best fit ellipse. The islands to include in the averaging can be selected by choosing a subset of set of islands of the summary_table by indexing on one or more variables. See data_analysis_examples for an example of using this function.


      Figure 9. Example of average displacement and traction vector fields produced by summarize_islands

    7. The circular islands described in this protocol and used in our work (Kaylan et al., 2018) have radial symmetry. Therefore, it was informative to analyze the traction a function of a single radial dimension as shown in Figure 10. The function summarize_islands_1D is provided to perform this analysis. Here, the XY position of each data point is converted to a radial coordinate, and the radial position is normalized by the measured radius of the island. The traction data is binned by radial coordinate, and a mean is taken. The data used for this analysis can also be selected using the summary table as discussed previously. The function also outputs the data table with the peak traction of each island amended to the relevant lines. See data_analysis_examples for an example of using this function.


      Figure 10. Tractions from 6 replicates of 4 conditions in one TFM dish plotted as a function of normalized radial coordinate. Dashed line shows standard deviation. In all conditions, traction peaks near the island periphery, where R = 1.

Recipes

  1. 2x ECMP Printing Buffer
    1. Add 164 mg sodium acetate and 37.2 mg EDTA to 5.9 ml dH2
    2. Once dissolved, add 50 µl warmed Triton X-100 and 4 ml glycerol. Vortex until combined
    3. Add approximately 80 µl of glacial acetic acid, titrating to adjust the pH to 4.8
    4. Store at 4 °C until arraying
  2. 2x Growth Factor (GF) Printing Buffer
    1. Add 105.5 mg sodium acetate and 37.2 mg EDTA to 6 ml PBS
    2. Once dissolved, add 0.1 g CHAPS and 3.8 ml glycerol
    3. Store at 4 °C until arraying
  3. Polyacrylamide Prepolymer Solution
    1. Polyacrylamide solutions are prepared according to the desired stiffness. An example set of stiffness recipes is adapted from (Wen et al., 2014)
      4 kPa Young’s modulus: 0.400 g acrylamide and 0.040 g bis-acrylamide
      13 kPa Young’s modulus: 0.600 g acrylamide and 0.045 g bis-acrylamide
      30 kPa Young’s modulus: 0.800 g acrylamide and 0.055 g bis-acrylamide
    2. Add appropriate amount of acrylamide and bis-acrylamide to 10 ml dH2O
    3. Vortex until the solution is clear. Filter solution with 0.2 µm syringe 
    4. Store solution at 4 °C for up to one month

Acknowledgments

We acknowledge the Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana–Champaign Core Facility for assistance with microscopy. Research reported in this publication was supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Number T32EB019944. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Competing interests

The authors declare that they have no competing financial interests.

References

  1. Butler, J. P., Tolic-Norrelykke, I. M., Fabry, B. and Fredberg, J. J. (2002). Traction fields, moments, and strain energy that cells exert on their surroundings. Am J Physiol Cell Physiol 282(3): C595-C605. 
  2. Dertinger, S. K., Jiang, X., Li, Z., Murthy, V. N. and Whitesides, G. M. (2002). Gradients of substrate-bound laminin orient axonal specification of neurons. Proc Natl Acad Sci U S A 99(20): 12542-12547. 
  3. Discher, D. E., Mooney, D. J. and Zandstra, P. W. (2009). Growth factors, matrices, and forces combine and control stem cells. Science 324(5935): 1673-1677. 
  4. Flaim, C. J., Chien, S. and Bhatia, S. N. (2005). An extracellular matrix microarray for probing cellular differentiation. Nat Methods 2(2): 119-125. 
  5. Hadden, W. J., Young, J. L., Holle, A. W., McFetridge, M. L., Kim, D. Y., Wijesinghe, P., Taylor-Weiner, H., Wen, J. H., Lee, A. R., Bieback, K., Vo, B. N., Sampson, D. D., Kennedy, B. F., Spatz, J. P., Engler, A. J. and Choi, Y. S. (2017). Stem cell migration and mechanotransduction on linear stiffness gradient hydrogels. Proc Natl Acad Sci U S A 114(22): 5647-5652. 
  6. Han, S. J., Oak, Y., Groisman, A. and Danuser, G. (2015). Traction microscopy to identify force modulation in subresolution adhesions. Nat Methods 12(7): 653-656. 
  7. Kane, R. S., Takayama, S., Ostuni, E., Ingber, D. E. and Whitesides, G. M. (1999). Patterning proteins and cells using soft lithography. Biomaterials 20(23-24): 2363-2376. 
  8. Kaylan, K. B., Berg, I. C., Biehl, M. J., Brougham-Cook, A., Jain, I., Jamil, S. M., Sargeant, L. H., Cornell, N. J., Raetzman, L. T. and Underhill, G. H. (2018). Spatial patterning of liver progenitor cell differentiation mediated by cellular contractility and Notch signaling. Elife 7: e38536.
  9. Kaylan, K. B., Kourouklis, A. P. and Underhill, G. H. (2017). A high-throughput cell microarray platform for correlative analysis of cell differentiation and traction forces. J Vis Exp (121). 
  10. Kourouklis, A. P., Kaylan, K. B. and Underhill, G. H. (2016). Substrate stiffness and matrix composition coordinately control the differentiation of liver progenitor cells. Biomaterials 99: 82-94.
  11. Kulkarni, A. H., Ghosh, P., Seetharaman, A., Kondaiah, P. and Gundiah, N. (2018). Traction cytometry: regularization in the Fourier approach and comparisons with finite element method. Soft Matter 14(23): 4687-4695. 
  12. Landauer, A. K., Patel, M., Henann, D. L. and Franck, C. (2018). A q-Factor-Based digital image correlation algorithm (qDIC) for resolving finite deformations with degenerate speckle patterns. Exp Mech 58(5): 815-830. 
  13. Linkert, M., Rueden, C. T., Allan, C., Burel, J. M., Moore, W., Patterson, A., Loranger, B., Moore, J., Neves, C., Macdonald, D., Tarkowska, A., Sticco, C., Hill, E., Rossner, M., Eliceiri, K. W. and Swedlow, J. R. (2010). Metadata matters: access to image data in the real world. J Cell Biol 189(5): 777-782. 
  14. Mertz, A. F., Che, Y., Banerjee, S., Goldstein, J. M., Rosowski, K. A., Revilla, S. F., Niessen, C. M., Marchetti, M. C., Dufresne, E. R. and Horsley, V. (2013). Cadherin-based intercellular adhesions organize epithelial cell-matrix traction forces. Proc Natl Acad Sci U S A 110(3): 842-847.
  15. Polacheck, W. J. and Chen, C. S. (2016). Measuring cell-generated forces: a guide to the available tools. Nature Methods 13(5): 415-423. 
  16. Romanov, V., Davidoff, S. N., Miles, A. R., Grainger, D. W., Gale, B. K. and Brooks, B. D. (2014). A critical comparison of protein microarray fabrication technologies. Analyst 139(6): 1303-1326. 
  17. Sabass, B., Gardel, M. L., Waterman, C. M. and Schwarz, U. S. (2008). High resolution traction force microscopy based on experimental and computational advances. Biophys J 94(1): 207-220.
  18. Schwarz, U. S. and Soine, J. R. (2015). Traction force microscopy on soft elastic substrates: A guide to recent computational advances. Biochim Biophys Acta 1853(11 Pt B): 3095-3104. 
  19. Tse, J. R. and Engler, A. J. (2010). Preparation of hydrogel substrates with tunable mechanical properties. Curr Protoc Cell Biol Chapter 10: Unit 10 16. 
  20. Wen, J. H., Vincent, L. G., Fuhrmann, A., Choi, Y. S., Hribar, K. C., Taylor-Weiner, H., Chen, S. and Engler, A. J. (2014). Interplay of matrix stiffness and protein tethering in stem cell differentiation. Nat Mater 13(10): 979-987.

简介

细胞微环境的组成和机械性能以及细胞向心力的分布会影响细胞的功能和行为。牵引力显微镜(TFM)提供了一种测量粘附细胞施加到表面的力的方法。文献中已经描述了许多TFM系统。广泛地讲,这些涉及在具有嵌入的荧光标记的柔性基底上培养细胞,所述荧光标记在细胞力松弛之前和之后成像。根据这些图像,计算出位移场,并且根据位移场,计算出牵引场。在这里,我们描述了一种TFM系统,该系统使用聚丙烯酰胺底物和微阵列点样仪在细胞外基质(ECM)蛋白或其他生物分子的各种组合上制造多细胞岛的阵列。具有自动镜台的显微镜用于在用去污剂裂解细胞之前和之后对每个细胞岛成像。使用一系列MATLAB脚本以半自动化方式分析这些图像,这些脚本会产生位移和牵引力场以及摘要数据。通过将微阵列与TFM分析的半自动化实现相结合,该协议可以评估底物刚度,基质组成和组织几何形状对高通量细胞力学行为的影响。
【背景】
细胞产生的力通过整合素传递至细胞外基质(ECM),并通过钙黏着蛋白传递至周围细胞。与这些力的传导相关的信号传导在调节增殖,分化,形态发生和体内平衡中起重要作用。此外,细胞力的大小和分布受微环境条件的影响,例如ECM组成,几何形状,底物刚度以及其他配体或药物的存在(Discher et al。,2009)。 。我们最近展示了ECM成分和底物刚度如何共同影响肝祖细胞的分化和收缩力(Kourouklis et al。,2016),并且细胞收缩力和牵引应力分布可以促进肝祖细胞的空间模式细胞分化(Kaylan等人,2018年)。研究微环境条件(例如ECM组成,几何形状,底物刚度以及其他配体或药物的存在)介导生物力学和生化反应的方式,需要工程化的系统来实现对环境的系统控制以及分析结果的方法行为。

在此协议随附的MATLAB代码中,使用公开可用的数字图像相关性(DIC)算法计算位移场(Landauer et al。,2018)。牵引力的计算是使用可用的傅立叶变换牵引细胞计数(FFTC)算法进行的(Sabass等,2008; Han等,2015)。选择这些方法是基于它们相对较低的计算成本和时间以及相对较少的所需用户输入。这支持了分析的高通量性质,并旨在为那些不将TFM作为核心能力的实验室提供“现成的”使用。但是,该代码旨在使用户能够根据用户和应用程序的需求以及计算资源来替代替代的位移或牵引场算法。

进行TFM通常涉及大量制备粘合剂基质,这需要相对大量的粘合剂蛋白,从而降低了对多种ECM蛋白或表面结合配体进行TFM的实用性。在这里,我们使用接触式印刷微阵列,该技术允许将多个ECM /配体组合(含重复样品)以低材料使用率包含在单个基材上(Flaim et al。,2005)。 TFM通常在单个细胞或随机分布的小菌落上实施。在微阵列系统中,我们可以评估多细胞岛的机械行为,在这些岛上,力会传递到基底以及邻近细胞,从而导致集体行为(Mertz et al。,2013)。此外,这些多细胞岛具有一致的尺寸和形状。这允许根据环境条件分析这些岛的平均机械性能。岛的直径可以通过使用不同大小的微阵列管脚进行调整,添加几何形状作为要研究的附加参数。提供的MATLAB代码包括识别岛边界并对齐来自复制岛的数据的技术,以分析这些平均行为。这些细胞微阵列可以使用免疫细胞化学方法进行平行测定,该方法已在其他地方进行了详细描述(Kaylan等,2017),以实现机械行为与表型行为的相关性。
我们的应用集中在机械行为的空间模式与肝祖细胞分化之间的关系。该方法可以应用于其他干细胞系统,特别是在空间模式和集体机械行为受到关注的地方。它也可以用于研究癌症的机械行为与微环境的关系,并进一步影响药物的扩散或反应。尽管此处我们使用接触式微阵列印刷,但该协议可以轻松地应用于其他具有微接触的微图案化系统,例如具有非接触式印刷的系统(Romanov等人,2014年)和聚二甲基硅氧烷(PDMS)压印的基材(凯恩(Kane)等人(1999)。在此应用程序中,我们利用阵列中的XY位置来了解该岛的状况。该协议可以扩展到其他系统,在这些系统中,细胞在其基质上的空间位置与实验变量或基质(例如具有刚性梯度(Hadden等,2017)或生物分子(Dertinger et al。,2002)。

关键字:牵引力显微镜, TFM, 多细胞岛, 微阵列, 基底刚度, 微环境, 机械生物学, 细胞力

材料和试剂

  1. 铝箔
  2. 钳子
  3. 移液器
  4. 1毫升注射器
  5. 6孔聚苯乙烯微孔板(Fisher Scientific,目录号:08-772-1B)
    注意:其他6孔微孔板可能也适用,但是35毫米玻璃底部培养皿要适合这些孔板,而不是全部6孔板都适合。
  6. 远红色荧光珠,0.2%v / v(Life Technologies,目录号:F-8816)
  7. 12毫米玻璃盖玻片(电子显微镜科学,目录号:72231-01)
  8. 384孔聚丙烯板(USA Scientific,目录号:1823-8400)
  9. 35毫米玻璃底盘,带13毫米孔和#1.5德国盖玻片(Cell E& G,目录号:GBD00002-200)
  10. 配体
    Fc重组DLL1,小鼠(R& D Systems,目录号:5026-DL-050)
    Fc重组DLL4,小鼠(AdipoGen,目录号:AG-40A-0145-C050)
    Fc重组JAG1,大鼠(R& D Systems,目录号:599-JG-100)
    人IgG(R& D Systems,目录号:1-001-A)
    蛋白质A / G(生命技术,目录号:21186)
    注意:细胞-细胞信号配体可以与ECM蛋白结合排列在表面上。较小分子在凝胶中的保留可能会有所不同,因此可能有必要将配体缀合到其他分子上。我们使用与蛋白A / G和IgG偶联的Fc重组缺口配体作为对照。如果使用这种策略,则配体应以6:1或更大的摩尔比与蛋白A / G结合。配体应按照制造商的建议进行重新配制,并储存在-80°C下。
  11. 细胞外基质蛋白
    胶原蛋白I,大鼠尾巴(EMD密理博,目录号:08-115MI),在4°C下保存
    人类胶原III(EMD密理博,目录号:CC054)
    IV型人类胶原蛋白(EMD Millipore,目录号:CC076)
    人纤连蛋白(西格玛奥德里奇,目录号:F2006)
    层粘连蛋白,小鼠(EMD密理博,目录号:CC095)
    注意:许多ECM蛋白都适合此过程。 ECM蛋白的选择取决于具体应用。在这里,我们列出了我们在各种研究中使用的那些,除非另有说明,否则均以1 mg / ml的浓度制备,并在-20°C下保存。
  12. 3-TPM(Sigma-Aldrich,目录号:440159-100ML)
    注意:暴露于空气中的3-TPM可能会对玻璃基板的硅烷化产生负面影响,应按照制造商的说明进行存储。这里描述的3-TPM包装在Sure / SealTM容器中,需要使用注射器和针头。
  13. NaOH(Sigma-Aldrich,目录号:415413-1L)
  14. 乙醇200证明(Decon Labs,目录号:UN1170)
  15. 丙烯酰胺(Sigma-Aldrich,目录号:A3553-100G)
  16. 双丙烯酰胺(Sigma-Aldrich,目录号:M7279-25G)
  17. Irgacure 2959(BASF Corporation,目录号:55047962)
  18. 甲醇(Sigma-Aldrich,目录号:179957-1L)
  19. 醋酸钠(Sigma-Aldrich,目录号:S2889-250G)
  20. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:ED-100G)
  21. 甘油(Sigma-Aldrich,目录号:M6145-25ML)
  22. 冰醋酸(Sigma-Aldrich,目录号:695092-500ML)
  23. 磷酸盐缓冲盐水(PBS)(Fisher Scientific,目录号:SH3001302)
  24. 3-[(3-氯氨基丙基)二甲基铵] -1-丙烷磺酸盐(CHAPS)(Sigma-Aldrich,目录号:C3023-1G)
  25. 右旋糖酐罗丹明(Life Technologies,目录号:D-1841)
  26. 100x青霉素-链霉素溶液(Fisher Scientific,目录号:SV30010)
  27. 牛血清白蛋白(Sigma-Aldrich,目录号:A2153-100G)
  28. 十二烷基硫酸钠(Fisher Scientific,目录号:BP166-100)
  29. 海卫一X-100(Sigma-Aldrich,目录号:X100-1L)
  30. 2个ECMP打印缓冲液(请参阅配方)
  31. 2x生长因子(GF)打印缓冲液(请参阅食谱)
  32. 聚丙烯酰胺预聚物溶液(请参阅食谱)

设备

  1. 真空干燥器(Fisher Scientific,目录号:08-642-5)
  2. OmniGrid微阵列分析仪(Digilab)
    注意:可以使用其他具有类似功能的微阵列仪。请按照制造商的说明操作阵列仪。
  3. 隐形或实心引脚(ArrayIt,目录号:SMP3或SSP015)
    注意:其他引脚类型可能适用于ArrayIt,SMP3可打印直径约为150μm的斑点。 SSP015引脚可打印直径约为600μm的斑点。选择适合您的应用的岛尺寸的引脚。
  4. 紫外线交联剂,UVP,CL-1000
  5. 涡流
  6. 荧光显微镜,蔡司Axiovert 200M,带有运行蔡司Zen图像采集软件的计算机
    注意:这里我们使用Zeiss Axiovert 200M,但是其他荧光显微镜也适用。显微镜应配备机械手平台并具有标记和查找位置的能力。由于TFM涉及活细胞成像,因此我们也建议环境控制(例如37°C和5%CO 2 )。可以直接从Zeiss专有格式(.czi)加载和读取元数据,但是由于它利用了Bio-Formats的工具(Linkert et al。 ,2010),文件只需加载很少或没有任何修改就可以加载大多数普通显微镜。

软件

  1. MATLAB版本2018b,Mathworks
  2. https://github.com/UnderhillLab/High-Throughtput-TFM 的MATLAB脚本< br /> 注意:提供的脚本旨在供具有基本MATLAB经验的人员使用,但是根据给定用户的额外需求调整脚本可能需要更高级的MATLAB专业知识。

程序

  1. 基板制造
    根据文献中的规程制备聚丙烯酰胺凝胶(Tse和Engler,2010; Wen et al。,2014)。
    1. 玻璃底盘的硅烷化。这使玻璃表面功能化,因此聚丙烯酰胺水凝胶将与玻璃表面共价结合。
      1. 向每个玻璃底盘的玻璃区域中加入250μl的0.2 N NaOH溶液。确保NaOH溶液覆盖整个玻璃区域,但不覆盖塑料表面,如图1A所示。
      2. 在轻轻摇动的振荡器上孵育1小时。
      3. 用去离子水(dH 2 O)冲洗盘子3次。
      4. 使用压缩的过滤空气干燥碗碟。将盘子在80°C的热板上的铝箔上烘烤,直到完全干燥。碟子过热可能会导致玻璃上的粘胶失效。
      5. 向9.8 ml 100%乙醇中添加0.2 ml甲基丙烯酸3-(三甲氧基甲硅烷基)丙酯(3-TPM)。
      6. 在每个培养皿的玻璃区域上涂抹100ul 3-TPM溶液。确保3-TPM溶液覆盖整个玻璃区域,但不覆盖塑料表面,如图1A所示。
      7. 在轻轻摇动的振荡器上孵育30分钟。
      8. 在每个培养皿中加入3毫升乙醇。在轻轻摇动的摇床上孵育5分钟。
      9. 除去乙醇。使用压缩空气擦干盘子。
      10. 将盘子在80°C的热板上的铝箔上烘烤,直到完全干燥。
        注意:在此阶段,硅烷化玻璃底盘可在黑暗干燥的地方存放长达一个月。


        图1.玻璃底盘硅烷化和凝胶制造。 A.适当覆盖NaOH和3-TPM溶液。 B.预聚物溶液的液滴。 C.盖玻片漂浮在预聚物液滴上。 D.倒盘以使珠子沉降。 E.紫外线照射后,装满去离子水的盘子。 F.去除盖玻片并脱水的盘子。

    2. 聚丙烯酰胺凝胶的制造。聚丙烯酰胺凝胶为TFM制备的一部分提供了可变形的水凝胶底物,供细胞结合。
      1. 根据所需的刚度准备聚丙烯酰胺预聚物溶液(配方3)。每个培养皿需要20μl的溶液。
      2. 向0.5 ml 100%甲醇中添加0.1 g Irgacure 2959(光敏性)以产生20%w / v Irgacure溶液。涡旋直至溶液澄清。
      3. 以9:1的比例合并预聚物溶液和Irgacure溶液。准备足够。
      4. 将珠悬浮液超声处理15分钟。
      5. 将小珠以1:500的比例添加到工作溶液中(即,将1μl小珠溶液与500μl工作溶液混合)。涡旋以确保完全混合。
      6. 将整个工作溶液在真空干燥器中脱气15分钟。
      7. 用移液管吸取20μl工作溶液到玻璃底盘的中央。避免液滴中出现气泡,如图1B所示。
      8. 小心地将12 mm盖玻片漂浮在液滴上。该溶液应散布液滴的边缘,如图1C所示。
      9. 重复其余的菜式。
      10. 倒置培养皿,如图1D所示,静置15分钟,以使珠子向表面迁移。
      11. 在UV交联剂中,将培养皿暴露于365 nM UVA中15分钟。
      12. 如图1E所示,用去离子水填充餐具。让我们静坐3-24小时。
      13. 用镊子小心地去除盖玻片。避免扭曲或剪切盖玻片,以免引起凝胶表面撕裂。
        注意:在dH 2 O中浸泡24小时后,使用干净的盖玻片并小心去除通常会成功去除盖玻片不会损坏凝胶。对于非常软的凝胶,可能需要进一步钝化盖玻片表面。策略包括使用精密的刮水器用Rain X®擦拭表面,或气相沉积二氯二甲基硅烷。
      14. 在热板上于50°C干燥凝胶,直到所有水从凝胶中蒸发为止。脱水的凝胶如图1A所示。凝胶盘在黑暗干燥的地方最多可以保存一个月。

  2. 芯片印刷
    该过程将一系列生物分子的圆形斑点印刷到聚丙烯酰胺基质上。这些阵列点是细胞将粘附形成圆形岛的地方。
    1. 如果需要,请准备合适的2倍打印缓冲区(配方1)
      注意:ECM蛋白印刷(ECMP)缓冲液适用于大多数ECM分子。生长因子(GF)打印缓冲液适用于其他种类的分子,例如生长因子或配体,低pH值可能会引起问题。
    2. 准备微阵列源板。
      1. 在384孔V底微孔板中,准备打印溶液,其中应包括等体积的2x打印缓冲液和生物分子溶液,浓度为目标浓度的两倍。对于许多ECM蛋白,250 µg / m是合适的浓度。最佳浓度将取决于分子,其保留及其功能。每个孔中的总体积应在5到15 µl之间。
      2. 在源板中,还应包括带有荧光标记的溶液,该溶液将用于传达阵列的方向以确定每种条件的位置。我们建议将罗丹明偶联的葡聚糖的终浓度为2.5 mg / ml。源极板配置将根据阵列器,引脚配置和所需的阵列布局而有所不同。
      3. 通过移液将各孔充分混合。注意避免产生气泡。将源微孔板以1,000 x g 的速度离心1分钟。源板可以立即使用,也可以在4°C下保存1天,直到制造微阵列。如果要存放,请用粘合密封剂盖住源板。
      4. 根据制造商的说明清洁并准备销钉。将清洁的针插入微阵列仪打印头。
      5. 使用制造商的软件准备微阵列仪和阵列程序。根据阵列器和所需的阵列布局,设置和编程将有所不同。应该设计程序,使阵列方向明确,并且每个阵列条件的位置是已知的,并且可以通过在任何方向上相对于荧光标记的位置来确定。图2提供了一个示例。
        注释:
        1. 所示的示例阵列布局设计用于打印4种条件的6个重复样本,这些重复样本由四个产生600μm直径岛的SSP015引脚同时打印。当使用产生较小岛的销时,可以按比例增加岛的数量和/或条件。
        2. 确保记录布局如何与排列的条件相对应。在每个条件之间包括使用水和二甲基亚砜(DMSO)进行洗涤的步骤,以防止残留和交叉污染。


        图2.带有葡聚糖若丹明标记的4种ECM条件阵列的示例布局。当岛和葡聚糖若丹明斑点可见时,无论旋转还是从上方或上方观察都可以辨别阵列的方向在下面。

      1. 打开加湿器,并确保将其调节到65%RH(无冷凝)。等待流变仪与设定点匹配。
      2. 将源板放在合适的适配器中的阵列器中。
      3. 在50°C下将水凝胶基质脱水15分钟。由于存在荧光珠,因此可以避光。&nbsp;
      4. 将碟子放入合适的适配器中。如果阵列仪可以安装标准多孔板,则6孔板适合盛放培养皿。
        注意:请参见“材料和试剂#5”下的注释。
      5. 开始阵列制造。经常检查湿度是否未降低到RH的65%以下(无冷凝)。
      6. 程序完成后,在室温和65%RH(非冷凝)下,将铺有铝箔的预制阵列存放一整夜。虽然可以看到阵列点,但在视觉上标记阵列的顶部或底部会有所帮助,因此在放置在显微镜上时可以知道方向。对于某些水凝胶和大头针组合,可能需要将阵列在环境温度和湿度下再存储两天,以确保阵列完全干燥。阵列在使用前最多可以存储7天。

  3. 在微阵列上接种细胞
    在这里,细胞从其正常培养条件转移到用于TFM的微阵列水凝胶基质上。
    1. 要对凝胶进行灭菌,请添加3 ml PBS和1%v / v青霉素/链霉素。暴露于UV C 30分钟。将青霉素/链霉素溶液换成细胞培养基。
    2. 按照细胞适当的程序收集和计数细胞。将细胞以合适的浓度重悬于培养基中进行接种。这取决于细胞类型,但可能在170 x 10 3 和7 x 10 5 细胞/ ml之间。在每个培养皿中加入3 ml细胞悬液。将培养皿在37°C和5%CO 2 中孵育2-24小时,或直至形成融合的细胞岛。可能需要针对您的细胞和应用优化播种密度和时间。每15-60分钟搅动盘子也可能有助于形成一致的,汇合的岛屿。
    3. 形成岛状物后,用3 ml预热的培养基冲洗阵列两次。在此阶段,添加任何实验性处理方法,例如生长因子或抑制剂。每1-2天更换一次培养基,直到进行TFM为止,或者按照您的细胞培养方案的建议,在每次交换时保持任何治疗浓度。图3显示了带有细胞岛的阵列的示例。


      图3.培养24小时后带有细胞岛阵列的35毫米玻璃底盘

  4. 牵引力显微镜的图像采集
    1. 准备1%v / v牛血清白蛋白(BSA)和1%v / v十二烷基硫酸钠(SDS)在PBS中的溶液。这将用于在图像获取过程中解离细胞。
    2. 显微镜检查过程将根据所使用的显微镜而有所不同,但以下步骤应大致适用。提供了一些其他详细信息,可与Zeiss Zen软件一起使用。显微镜应具有机械手平台和可提供孵化和湿度控制的腔室,这些腔室已适当设置并使其达到平衡。将一盘放在倒置荧光显微镜的平台上。使用阵列制作后提供的标记来放置碟子,使阵列的XY轴与显微镜平台的轴大致对齐。将碟子固定在适当的位置,以防止其在平台移动时移动。
    3. 使用适当的荧光通道,找到并标记(在软件中)荧光阵列标记的XY位置。在Zeiss Zen Blue 2中,这是在“获取”部分中完成的,方法是选中图块框并为每个岛添加一个位置,如图4所示。


      图4. Zeiss Zen Blue 2环境。要标记位置,请在采集中标记Tiles并输入高级设置。设置各个岛的位置,如黄色标记所示。可以使用验证位置工具在各个位置之间移动以设置Z位置,而无需更改XY位置。

    4. 使用相衬或明场,定位并标记细胞岛的XY位置。只要图像可以在后续分析中提供细胞岛的边界,任何一种成像方式都可能适用。分别找到并保存每个岛的Z平面焦点。示例图像相位对比在图5A中示出。验证位置工具可用于在各个位置之间移动以设置Z位置,而无需更改XY位置。可以保存孤岛的已保存XY位置,并将其重新用于以后的菜肴。
    5. 开始对细胞岛的相衬图像进行自动成像。使用合适的名称保存该文件,该名称应注明实验细节以及它是相衬图像。
    6. 切换到适合珠子的荧光通道。分别找到并保存每个岛下方凝胶顶部表面的Z平面焦点。注意避免改变XY位置。使用适当的名称保存该文件,该名称应标明实验详细信息,并且它是解离前的映像。图5B中显示了示例图像。


      图5.来自TFM数据收集的示例图像。 A。蜂窝岛的相位合同图像。图像应足够清晰,以定义岛的边界。 B.在岛下的基底表面上的珠子的荧光图像。比例尺:200μm。

    7. 在解离前的珠子图像的荧光通道中开始自动成像。使用相同的实验详细信息保存该文件,并将其保存为解离前的图像。
    8. 小心地向培养皿中加入150 µl SDS溶液,注意不要碰撞或移动培养皿。使用相衬通道监控细胞岛的解离。等到岛完全与基板分离后,此时在相衬通道中观察到的岛位置大部分应该是空白的。
      注意:某些细胞可能需要添加更多的SDS溶液或更高的浓度。此外,也可以改用Triton-X。
    9. 返回岛位置,并使用荧光通道检查表面顶部的珠子是否仍在焦点上。如果珠子由于细胞引起的变形的松弛而移出了平面,请校正并保存焦平面的Z坐标,但不要更改XY位置。重复对岛进行自动成像,以捕获珠子的解离后图像。珠的位移将可能难以辨别,这意味着解离后的荧光珠图像应看起来与解离前的图像非常相似。使用合适的名称保存该文件,该名称应标明实验细节以及它是解离后的图像。
    10. 对每道菜重复步骤D2-D9。

数据分析

  1. 处理图像以估计位移场并计算牵引力
    注意:上一部分收集的图像可以导出并与可用的TFM代码包一起使用。在这里,我们提供了已实现的TFM代码,以下部分将说明其用法。 TFM和分析旨在与具有相同几何形状的细胞阵列一起使用,以能够评估相同条件的重复。这些脚本允许用户生成TFM数据并创建基本可视化。对用户特定应用程序的数据进行进一步的处理和分析可能需要生成其他脚本。

    1. 确保提供的MATLAB代码已保存到适当的位置。导航到该目录已保存的文件夹。
    2. 确保所有图像文件均已保存或转移到计算机上可用的文件夹中,以用于分析。
    3. 在命令窗口中,运行无输入的功能 run_island_tfm 。
    4. 系统将提示您选择带有相衬图像的文件。导航到并选择带有相衬图像的文件。
    5. 一个新的文件选择窗口将在与所选文件相同的目录中打开。现在,选择带有解离前荧光图像的文件。最后,在出现提示时,选择解离后图像。
    6. 将出现一个GUI,用于输入图像文件的实验信息。该GUI如图6所示。图形面板显示了所收集的每个图像的XY位置。数字对应于图像在图像文件中的索引方式。输入“数据文件名”字段上方列出的所有相关实验信息。像素大小将从图像的元数据中读取,但可以更改。 “阵列条件的数量”是指成像阵列中不同条件的数量。对于此协议中的示例布局,有4个数组条件。此数字用于将岛屿分组到其条件中。


      图6.用于输入实验和皿信息的GUI。“图表”面板显示每个岛的XY位置,并以文件中岛的索引标记。图表标题是图像文件名。像素大小是从图像元数据中提取的,但是可以手动设置。所有其他字段均由用户设置。

    7. 完成这些字段后,单击“设置信息”。这将使用输入的信息填充“数据文件名”字段,该信息将作为输出文件的文件名。可以手动更改此字段。将出现两个附加字段。在“条件1”字段中输入第一个条件的名称。在“条件为1的岛屿”字段中输入分配有此条件的所有岛屿的数字作为数字列表。单击“设置条件”。如果还有其他条件,请对其余每个条件重复这些步骤。设置完所有条件后,将显示“完成”按钮。确认所有实验信息正确无误,然后单击“完成”。将分析在此步骤中分配了条件的所有孤岛。要仅分析某些岛屿,请将条件数量设置为要分析的岛屿所代表的数量条件,并仅在“有条件的岛屿”字段中列出这些岛屿。
    8. 现在,脚本将循环遍历条件和孤岛。它将首先运行脚本以更正解离前后的帧偏移。
      注意:正确的移位功能是消除图像之间的全帧移位,这是由于收集图像之间的阶段漂移所致。这是通过在距离没有预期变形的像元足够远的图像区域上执行数字图像关联来完成的。该脚本会自动选择图像四个角附近的四个区域。要手动选择这些区域,请在frame_shift函数调用中将“ n”更改为“ y”。
      现在,GUI似乎将有助于在单元岛周围绘制边界。此GUI如图7所示。软件将尝试在单元岛周围绘制边界,并以红色绘制。幻灯片可用于调整跟踪的参数,这将导致跟踪实时重新运行。您也可以通过在文本框中输入数字,然后单击“重新运行”按钮来更改这些参数的值。或者,您可以通过单击“手动绘制”按钮来手动绘制边界。在岛上单击鼠标左键,直到边界被封闭。在边界内双击以创建形状。为避免出现尖角,绘制多边形后,使用“模糊”字段中的当前值进行模糊和舍入。为避免这种舍入,请将此字段设置为1。手动绘制功能可用于跟踪多个区域。要重置手动边界,请单击“清除边界”,或单击“重新运行”以重复自动跟踪。对边界满意后,单击“完成”。


      图7.用于选择单元岛边界的GUI。可以使用滑块设置参数,这些滑块将实时更新边界,或者输入值并单击Rerun。您也可以手动绘制边界。

    9. 这将带来下一个岛屿。对选择进行分析的文件中的所有岛重复此过程。
    10. 当完成每个岛的边界跟踪时,将保存数据。然后,程序将移动以计算每个岛的位移和牵引力场。根据计算机上的图像数量,图像大小和处理器,此过程最多可能需要几个小时。在每个孤岛之后保存数据,以在分析期间出现问题的情况下限制数据丢失。数据保存在“数据输出”文件夹中。
    11. 大部分的计算时间是位移场估计。要在已经确定了边界并计算了位移场的文件上使用不同设置重新运行牵引力场计算,请运行命令 run_island_tfm (' rerun ')。系统将提示您选择要重新分析的数据文件。

  2. 查看数据并生成摘要
    1. 在提供的代码中,将输出与分析期间建立的文件名一起保存在“数据输出”文件夹中。要浏览单个文件中的数据,可通过双击已浏览的当前文件夹或使用加载功能将数据加载到工作区中。
    2. 这会将单元格数组“ all_cell_data”加载到工作空间中。每个单元格对应于分析中的单个岛。该输出旨在为用户提供对所有相关数据的轻松访问,以供用户在MATLAB中进行浏览和分析,或导出至其他程序以进行适合其应用程序的分析。表1提供了存储在输出文件中的数据的组织。可以使用点格式的structName.fieldName来访问数据结构元素。
      注意:请参见 https://www.mathworks.com/help/matlab/matlab_prog/access-data-in-a-structure-array.html 有关访问数据的更多信息在结构中。
    3. 提供了一组功能来查看和分析数据。 data_analysis_examples 中提供了使用这些功能的示例代码。要查看单个岛屿的搜索结果,请使用 v iew_one_island 。请参阅代码以获取更多文档。输出示例如图8所示。


      图8. view_one_island 的示例输出。显示岛屿信息。向量场绘制在位移场(以μm为单位)和牵引场(以Pa为单位)的大小上。岛边界以黑色绘制。还显示相衬图像,边界以黄色绘制。最佳拟合的椭圆以长轴和短轴显示为红色。

    4. 通过使用岛的边界,脚本可以找到最合适的椭圆。该椭圆可用于对齐比较具有相同几何形状的多个岛。
      注意:提供的脚本假定这些岛可以用椭圆表示。通过更改识别旋转中心和旋转角度的方式,可以使其适应其他几何形状。
      使用view_one_island_rotandcen查看根据最佳拟合椭圆居中和旋转的数据。
    5. 提供功能collect_island_data可以从多个TFM运行的多个岛中收集数据。此函数的输出是一个表格,其中包含从文件中加载的每个岛的数据和信息(请参见表1),以及一个包含位移和牵引力数据的结构,该结构根据汇总表的“ summ_ind”字段建立索引。表可以导出到excel或类似的文件。在没有输入的情况下运行该功能时,系统将提示您选择要加载和合并的数据文件。

      表1.数据输出文件的结构和保存的数据的描述
      <身体> class="“" ke-zeroborder”="" bordercolor="“#000000”" style="“" width:800px;”="" border="“" 0”="" cellspacing="“" cellpadding="“" 2”=""> 数据字段


      说明
      cell_info。
      年轻

      底材的杨氏模量

      点数

      底物的泊松比

      像素大小

      原始图像像素的大小,以微米为单位

      实验

      实验说明

      date_ID

      实验或运行的标识符

      可溶

      添加到盘子中的可溶性因子或处理方法

      dish_num

      如果有特殊情况的复制品,则为菜品的标识,例如

      时间

      播种和TFM数据收集所需的时间

      arrayed_condition

      ECM或阵列中孤岛的其他条件

      X位置

      阵列中岛的X位置

      Yposition

      阵列中岛的Y位置

      图像编号

      原始文件中的图像索引

      文件

      包含原始相位/明场图像的文件

      dm

      位移/牵引力输出的最终间距(以像素为单位)




      图像。
      图像{1}

      离解前的荧光图像

      图像{2}

      解离后的荧光图像

      图像{3}

      相位/明场图像




      单元格边界。
      蒙版

      在岛屿边界内确定的区域的遮盖物

      boundary_points
      包含所跟踪的每个边界的XY坐标的单元格

      cny

      用于跟踪边界的灵敏度因子

      sigma

      用于跟踪边界的模糊系数

      di

      用于跟踪边界的侵蚀因子

      ellipse_fit。
      phi&nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;
      合适的椭圆相对于水平轴的旋转角度


      X_center
      以非旋转椭圆的X轴为中心

      Y_center 以非旋转椭圆的Y轴为中心 &nbsp; &nbsp; &nbsp;&nbsp; X0
      以旋转椭圆的X轴为中心


      Y0
      以旋转椭圆的Y轴为中心


      主轴_span>
      椭圆的长轴(以像素为单位)


      minor_axis&nbsp;
      椭圆的小轴(以像素为单位)

      rotated_boundary_points&nbsp; &nbsp; &nbsp; &nbsp; &nbsp;

      包含根据拟合椭圆跟踪,旋转和居中的每个边界的XY坐标的单元格




      cell_displacements。&nbsp; &nbsp; &nbsp; &nbsp;&nbsp;
      原始位移。 原始位移{1}&nbsp; &nbsp; &nbsp;&nbsp; 原始估算位移字段的X分量(以像素为单位)


      原始位移{2}&nbsp; &nbsp; &nbsp; &nbsp; &nbsp;
      原始估算位移场的Y分量(以像素为单位)


      原始位移{3}&nbsp; &nbsp;&nbsp;
      原始估算位移的幅度(以像素为单位)

      位移。
      位移{1}
      位移场的X分量,单位为微米


      位移{2}
      位移场的Y分量,单位为微米


      位移{3}&nbsp; &nbsp; &nbsp; &nbsp;
      位移场的幅度以微米为单位

      旋转位移

      已使用合适的椭圆旋转并居中的位移




      cell_tractions。
      交易。
      牵引力{1}
      牵引力的X分量


      牵引力{2}
      以Pa为单位的牵引力的Y分量


      牵引力{3}
      Pa的牵引量

      RMS

      牵引力的均方根

      RMS_int

      单元格岛边界内牵引场的均方根

      Strain_Energy

      应变能

      rotated_tractions

      使用适合椭圆旋转并居中的牵引力

    6. collect_island_data 函数的输出可以进一步用于创建摘要数据。函数 summarize_islands 绘制并输出平均位移和牵引力场,示例参见图9。此功能基于最佳拟合椭圆使用来自每个岛的对齐数据。可以通过对一个或多个变量建立索引来选择summary_table的一组孤岛的子集,从而选择要包含在平均值中的孤岛。有关使用此功能的示例,请参见 data_analysis_examples 。


      图9.由 summarize_islands
      产生的平均位移和牵引矢量场的示例
    7. 该协议中描述并在我们的工作中使用的圆形岛(Kaylan等,2018年)具有径向对称性。因此,如图10所示,分析单个径向尺寸的牵引力很有帮助。提供了功能 summarize_islands_1D 来执行此分析。在此,每个数据点的XY位置都转换为径向坐标,并且该径向位置通过测得的孤岛半径进行归一化。牵引力数据通过径向坐标进行分类,并取平均值。如前所述,还可以使用汇总表选择用于此分析的数据。该功能还输出数据表,并将每个孤岛的峰值牵引力修改为相关行。有关使用此功能的示例,请参见 data_analysis_examples 。


      图10.绘制一个TFM皿中4个条件的6个重复样本的牵引力,作为归一化径向坐标的函数。虚线表示标准偏差。在所有情况下,牵引力在孤岛边缘附近达到峰值,其中R =1。
    8. 菜谱

      1. 2个ECMP打印缓冲区
        1. 向5.9 ml dH 2 O中添加164 mg乙酸钠和37.2 mg EDTA。
        2. 溶解后,加入50 µl温热的Triton X-100和4 ml甘油。涡旋直到合并
        3. 加入约80 µl冰醋酸,滴定以将pH调节至4.8
        4. 储存在4°C直到排列
      2. 2x生长因子(GF)打印缓冲液
        1. 在6 ml PBS中加入105.5 mg乙酸钠和37.2 mg EDTA
        2. 溶解后,加入0.1 g CHAPS和3.8 ml甘油
        3. 储存在4°C直到排列
      3. 聚丙烯酰胺预聚物溶液
        1. 根据所需的硬度制备聚丙烯酰胺溶液。一组示例性的刚度配方改编自(Wen et al。,2014)
          4 kPa杨氏模量:0.400克丙烯酰胺和0.040克双丙烯酰胺
          13 kPa杨氏模量:0.600克丙烯酰胺和0.045克双丙烯酰胺
          30 kPa杨氏模量:0.800克丙烯酰胺和0.055克双丙烯酰胺
        2. 在10 ml dH 2 O中添加适量的丙烯酰胺和双丙烯酰胺
        3. 涡旋直至溶液澄清。用0.2 µm注射器过滤溶液
        4. 将溶液在4°C下保存最多一个月

      致谢

      我们感谢伊利诺伊大学厄本那-香槟分校的卡尔·R·沃斯基因组生物学研究所在显微镜检查方面的协助。该出版物中报道的研究得到了美国国立卫生研究院国家生物医学成像和生物工程研究所的资助,编号为T32EB019944。内容仅由作者承担,并不一定代表美国国立卫生研究院的正式观点。

      利益争夺

      作者宣称他们没有竞争的财务利益。

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引用:Berg, I. C. and Underhill, G. H. (2019). High Throughput Traction Force Microscopy for Multicellular Islands on Combinatorial Microarrays. Bio-protocol 9(21): e3418. DOI: 10.21769/BioProtoc.3418.
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