Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

All animal procedures were approved by the University of British Columbia Animal Care Committee.

1. Expressing GFP-fusion proteins in A20 B-lymphoma cells

1. Culture A20 cells in complete RPMI medium (RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 µM 2-mercaptoethanol, 2 mM glutamine, 1 mM pyruvate, 50 U/mL penicillin, and 50 µg/mL streptomycin) at 37 °C in a tissue culture incubator with 5% CO₂.
2. Prepare the transfection reagent (see the Table of Materials) according to the manufacturer's instructions.
   NOTE: This protocol has been optimized for the specific reagents and transfection protocol described in the Table of Materials. Other transfection reagents that we have tried have not yielded sufficient transfection efficiency.
3. Count the cells using a hemocytometer. Centrifuge 2.5 x 10⁶ A20 cells (for each transfection) for 5 min at 525 x g. Resuspend the cells in 100 µL of transfection reagent containing 1-2.5 µg of plasmid DNA. For DNA encoding CLIP-170-GFP²³, use 2.5 µg per transfection. Mix gently.
4. Transfer the cells to a well of a 6-well plate, and adjust the volume to 2 mL with pre-warmed complete RPMI medium. Culture the cells for 18 h at 37 °C to allow protein expression.

2. Isolating Primary Mouse B Cells and Activating Them with LPS

1. Use sterilized surgical tools to remove the spleen from a mouse, following protocols approved by the institution's Animal Care Committee.
2. In the tissue culture hood, place a sterile 70-µm cell strainer into a 35-mm tissue culture dish containing 3 mL of room temperature sterile PBS. Use the rubber part of the plunger from a 5-ml syringe to crush the spleen through the cell strainer.
3. Pipet the single cell suspension of splenoctyes into a sterile 15-mL tube and centrifuge at 525 x g for 5 min.
4. Use a magnetic bead-based B cell isolation kit (see Table of Materials) to obtain a highly enriched population of B cells.
5. Resuspend the B cells to 3 x 10⁶/mL in complete RPMI medium supplemented with 2.5 µg/mL LPS plus 5 ng/mL B cell activating factor (BAFF; a survival cytokine).
6. Culture the cells for 6 hr at 37 °C.

3. Coating Glass Coverslips with Anti-Ig Antibodies

1. Prepare the antibody solution for coating the coverslips. Dilute the stock solution of goat-anti-mouse Ig antibodies into room temperature sterile PBS to make a 12.5 µg/mL solution; 400 µL of antibody solution is required for each coverslip.
   NOTE: Use goat anti-mouse IgG for A20 cells; use goat anti-mouse IgM for primary B cells. Goat IgG antibodies do not bind well to mouse Fc receptors. However, to avoid any potential Fc receptor binding, one can use F(ab) or F(ab')₂ fragments of anti-mouse IgG or anti-mouse IgM antibodies.
2. Dip a #1.5 18-mm round glass coverslip in 100% methanol and let it dry completely (~10 min).
3. Using forceps, place the dried coverslip into a 12-well tissue culture plate and pipet 400 µL of the 12.5 µg/mL antibody solution (5 µg total; 2 µg/cm²) onto the coverslip so that it forms a bubble at the center of the coverslip and spreads to the edges.
4. Be careful to cover the entire coverslip, but do not allow the antibody solution to extend past the edge of the glass coverslip and onto the tissue culture plastic. Incubate at room temperature for 30 min.
5. Wash the coverslips by pipetting 1 mL of sterile PBS into the well and subsequently aspirating out the solution. Repeat two more times to remove unbound antibodies.
6. Add 1 mL of sterile PBS to the well containing the antibody-coated coverslip until cells are ready to be added.
   NOTE: The coverslips can be kept at room temperature, covered with PBS, for use on the same day.

4. B Cell Spreading on Anti-Ig-Coated Coverslips

1. Prepare modified HEPES-buffered saline (mHBS): 25 mM HEPES, pH 7.2, 125 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM Na₂HPO₄, 0.5 mM MgSO₄, 1 mg/mL dextrose, 2 mM glutamine, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol). Filter sterilize this solution and store at 4 °C.
2. On the day of the experiment, make 50 mL of mHBS with 2% FBS (mHBS-FBS) by adding 1 mL of heat-inactivated FBS to 50 mL mHBS. Warm the mHBS-FBS to 37 °C before use.
3. Centrifuge the transfected A20 cells or the primary B cells that had been cultured with BAFF plus LPS at 525 x g for 5 min.
4. Resuspend the cells in 1 mL of mHBS-FBS, count the cells using a hemocytometer, and dilute the cells to 2 x 10⁵ cells/mL in mHBS-FBS.
5. Using forceps, transfer the coverslip from the tissue culture plate to a piece of paraffin film.
6. Add 250 µL of B cells (5 x 10⁴ cells) to each coverslip.
7. Incubate the coverslips at 37 °C for 15 min in the dark to allow the B cells to spread over the anti-IgG-coated surface.

5. Fixing and Immunostaining the Cells

1. Prepare the fixation solution by diluting the 16% paraformaldehyde stock solution and the 50% glutaraldehyde stock solution in room-temperature distilled water to yield final concentrations of 3% paraformaldehyde and 0.1% glutaraldehyde. Prepare the fixation solution on the day it is to be used and discard any excess.
   CAUTION: The paraformaldehyde and glutaraldehyde stock solutions should only be used in a chemical fume hood. Follow the instructions on the material safety data sheet (MSDS).
2. Prepare the permeabilization/blocking buffer (3% BSA and 0.1% Triton X-100 diluted in PBS). Filter sterilize this solution and store at 4 °C. On the day of the experiment, warm the required amount to room temperature.
3. Prepare the staining buffer (1% BSA and 0.1% saponin in PBS). Sterilize this solution using a 0.2 µm filter and store at 4 °C. On the day of the experiment, warm the required amount to room temperature.
4. Pipet 350 µL of the fixation solution onto each coverslip, ensuring that the surface is completely covered. Incubate at room temperature for 10 min in the dark.
5. Remove the fixation solution from the coverslip by carefully aspirating from the edge of the coverslip using a micropipet.
   CAUTION: The fixation solution should be discarded as hazardous chemical waste.
6. Wash the coverslips once with 500 µL of permeabilization/blocking buffer. Pipet off the liquid.
7. Permeabilize and block the cells by adding 250 µL of permeabilization/blocking buffer to each coverslip, and incubating for 10 min at room temperature in the dark.
8. Dilute the rabbit anti-tubulin antibody 1:100 in staining buffer.
9. Aspirate off the permeabilization/blocking buffer from the coverslips, and add 50 µL of the diluted anti-tubulin antibody solution to each coverslip. Incubate at room temperature for 30 min in the dark.
10. Prepare the secondary antibody/actin stain solution by diluting both the Alexa Fluor 532-conjugated goat anti-rabbit IgG secondary antibody and Alexa Fluor 568-conjugated phalloidin 1:100 in staining buffer.
11. Wash the coverslips 3 times to remove excess primary antibody by adding 500 µL of staining buffer and then aspirating it.
12. Add 50 µL of the secondary antibody/actin stain solution to each coverslip, and incubate for 30 min at room temperature in the dark.
13. Wash the coverslips 3 times to remove excess secondary antibody by adding 500 µL of staining buffer and then aspirating it.
14. Add 10 µL of mounting reagent to a microscope slide. Mount the coverslip onto the microscope slide with the cell side down.
    NOTE: An "anti-fade" mounting medium (see Table of Materials) is strongly recommended to preserve the fluorescence intensity of GFP fusion proteins. Avoid the use of mounting reagents containing DAPI, which can interfere with the imaging of the fluorophores when using the STED laser.
15. Allow the slides to dry overnight in the dark. Image the slides the next day.
    NOTE: Imaging the slides as soon as the coverslip is dry is strongly recommended, as the GFP fluorescence will decrease over time.

6. Imaging Using the STED Microscope

NOTE: Please note that all software steps described below are specific to the microscope and software we used (see the Table of Materials). The steps and settings will need to be adjusted if imaging is performed using a different microscope/software.

1. Turn on the STED microscope, activate the lasers and epifluorescence illumination lamp, and start up the microscope software.
2. Set the parameters for the STED depletion lasers.
   NOTE: This will depend on the specific microscope being used and the lasers that it is equipped with. Some recommended settings are white light (WLL) 70%; 592 nm laser 80%; 660 nm laser 80%.
3. Activate the STED settings and align the STED laser beams using the 100X objective.
4. Using the eyepiece, focus the sample and select a cell with moderate GFP expression.
   NOTE: Low GFP expression will not be visible because STED reduces the emission intensity. Conversely, high expression of CLIP-170-GFP induces the spontaneous formation of large clusters, which does not reflect the normal microtubule plus-end protein complex morphology and distribution. Selecting a cell with moderate GFP expression is essential for accurately imaging the cytoskeletal structures at the antigen contact site.
5. Zoom into the selected cell and choose the region of interest to be imaged. Adjust the focus so that the x-y plane of the B cell that is closest to the coverslip is in focus.
   1. To do this, move the objective progressively closer to the coverslip so that the B cell cytoskeletal structures come into focus and then go out of focus. Then gradually move the objective in the opposite direction until the B cell cytoskeletal structures first come back into focus.
6. Optimize the settings including the laser power, excitation beam, and detector range. Start with the settings recommended by the manufacturer's instructions. Then make adjustments as needed to optimize the signal for the samples. Image all samples to be compared to each other with the same settings.
7. Under the "acquire" tab of the software, set the image acquisition to sequential frame acquisition in the lower left "Sequential Scan" panel by checking the option "between frames".
8. Set the acquisition sequence.
   Note: We acquire the GFP fluorescence first in order to avoid degradation of the GFP signal.
   Depending on the combination of fluorophores that are used in addition to GFP, imaging the longer wavelength fluorescence signal first may yield better results. However, the order in which the fluorophores or fluorescent proteins are subjected to stimulated emission depletion and imaged should be optimized to obtain the strongest fluorescence signals and best resolution.
9. Select and set the STED laser power for each fluorophore under the "Acquire" tab of the software, and use the slider bar for either the 592-nm or the 660-nm STED laser to adjust the laser power. Adjust the power for the 592-nm depletion laser for GFP and for Alexa Fluor 532. Adjust the power for the 660-nm depletion laser for Alexa Fluor 568.
10. To increase the resolution and reduce the background signal, go to the "Acquisition" settings under the "Acquire" tab of the software, increase the line and/or frame averaging by selecting a value greater than 1 using the drop-down menu for each option.
11. Also, acquire the fluorescent signal using time-gated STED by checking the gating option for the fluorophore under the "Acquire" tab, and specifying values for time-gating (see note below). Increase the STED laser power to enhance the resolution, though this will also increase photobleaching of the fluorophores.
    NOTE: The gating time will need to be optimized for the microscope, sample, and fluorophores being used. A time gating of 0.3 to 6 ns is recommended. After fixation, GFP is particularly sensitive to high laser power. To reduce the photobleaching of GFP, time gating should be applied and the STED laser power should be reduced.
12. To capture 3D STED images, use the slider to change the point spread function (PSF) to "3D STED".
13. Manually acquire multiple images to ensure reproducibility. Deconvolve the images (see Figure 1) using a deconvolution software package⁴¹(see Table of Materials).