Real-time Live Imaging of T-cell Signaling Complex Formation

1. T-cell Transfection

1. Prepare the activated Amaxa Nucleofector solution by mixing the kit’s “Supplement” solution to the Nucleofector Solution. The activated solution may be stored at 4 °C for up to three months.
2. Grow Jurkat T cells in culture medium [10% fetal calf serum (FCS), 1% Penicillin-Streptomycin solution, and 2 mM L-glutamine in RPMI]. Optimally, cells should be used 1-2 weeks after thawing. Using cell cultures in logarithmic growth is crucial for high transfection efficiency and cell survival. Alternatively, with minor modifications, this protocol can be used with primary T cells, as previously described ⁸. It should be emphasized that as the duration of the activation process of primary T cells is longer than that of Jurkat T cells (~30 min), a microscope stage mounted incubation system must be used to maintain CO₂, humidity levels and temperature throughout the activation process.
3. Collect 5 × 10⁶ log-phase T cells per transfection into a 15-ml tube.
4. Centrifuge cells for 7 min at 400 × g at room temperature.
5. During centrifugation, prepare a 6-well plate. Fill one well for each transfection with 5 ml culture medium, and pre-incubate at 37 °C.
6. Collect the tubes from the centrifuge and discard the supernatant. Resuspend the cell pellet in 1 ml RPMI.
7. Centrifuge cells for 7 min at 400 × g at room temperature.
8. During centrifugation, prepare the transfection solution. In one well of a 96-well plate, mix activated 100 μl Amaxa Nucleofector solution prepared in step 1 with 5 μg DNA of the plasmid of interest encoding a fluorescently tagged protein. Optimally, DNA concentration should be greater than 1 μg/ml in order to avoid diluting the transfection solution.
9. Mix well by gentle pipetting.
10. After centrifugation of the cells, carefully remove all the supernatant while not disturbing the pellet.
11. Pipette the DNA/transfection solution twice.
12. Add the DNA/transfection solution to the pellet.
13. Transfer the cells to the Amaxa cuvette.
14. Insert the cuvette into the Amaxa Nucleofector device.
15. Use the H-10 Amaxa transfection setting for electroporation (for Jurkat ) or T-23 (for primary T cells).
16. Remove the 6-well plate from the incubator.
17. Following electroporation, carefully transfer the supernatant with the cell suspension to the 6-well plate using a Pasteur pipette. A pellet consisting of cell debris may form. Avoid transferring the pellet.
18. Incubate the cells for 24 hr at 37 °C.
19. Transfer 2.5 ml of cell suspension from each well into a new well. Add 2.5 ml warm culture medium (described in step 2) to each well.
20. After 72 hr, transfer the cells to a sterile tube and centrifuge for 7 min at 400 × g at room temperature. Discard the supernatant and replace it with culture medium supplemented with the mammalian cell selection antibiotic appropriate for the plasmid used (apply a concentration suitable for the antibiotics used; we used G418 and/or Hygromycin in concentrations of 1.3 mg/ml and/or 0.2 mg/ml, respectively). Culture the cell suspension in a fresh well. Alternatively, cells can be transfected transiently and imaged 48 hr after transfection. To prepare transiently transfected cells, co-transfect the cells with the two plasmids simultaneously at step 8, continuing normally to step 20, and then skip directly to step 23. Note that under this scenario, the cells should be used immediately, and generally their fluorescence intensity is heterogeneous and not necessarily high.
21. Grow cells with culture medium supplemented with the appropriate selection antibiotic. Split the cells and supplement them with antibiotic-supplemented culture medium as needed.
22. After 2 weeks, verify successful transfection with the use of FACS (fluorescence-activated cell sorting) as follows:
23. Collect 10⁶ transfected cells, 10⁶ mock transfected cells (negative control), and 10⁶ cells that are known to stably express the fluorescently tagged protein. If cells are transfected with more than one fluorescent protein (see step 27), use multiple, singly tagged cell lines as positive controls.
24. Centrifuge cells for 7 min at 400 × g at room temperature.
25. Discard the supernatant. Resuspend cells in 500 μl FACS buffer (PBS w/o Ca²⁺ and Mg²⁺, 5% FCS, 0.05% sodium azide).
26. Use an analytical FACS to verify cellular fluorescence. Compare fluorescence of the transfected cells to that of the negative and positive controls.
27. To transfect the cells with an additional, fluorescently tagged protein, repeat steps 3-26 using the transfected cells.
28. Cells displaying high levels of fluorescence should be used. Optimally, at least 50% of the cells in the culture should be highly fluorescent. Cell fluorescence may be increased by cell sorting via FACS.

Any additional tagged proteins must be fused to different fluorophores and be encoded on plasmids encoding for different selection antibiotics. Selection medium for cells transfected with more than one plasmid should be supplemented with all the appropriate antibiotics.

2. T-cell Spreading Assay Preparation

1. Use Lab-Tek II German coverglass system 4 chamber slides. Make sure not to accidently scratch the inner surface of the chambers, e.g. with a pipette tip, during preparation or use.
2. Prepare 50-ml slide preparation solution: Take 4.6 ml of 12 M (37%) HCl, add 38.5 ml 100% ethanol, and 6.9 ml doubly distilled water.
3. Fill each chamber of the slide with 500 μl slide preparation solution.
4. Incubate at room temperature for 10 min.
5. Aspirate the chambers, drying them completely.
6. Incubate the uncovered slides for 1 hr at 45 °C till fully dry.
7. Prepare 0.01% poly-L-lysine solution by diluting 0.1% poly-L-lysine (Sigma-Aldrich) in double distilled water.
8. Apply 500 μl to each chamber.
9. Incubate the slide for 15 min at room temperature.
10. Aspirate the chambers, drying them completely.
11. Incubate for 3 hr at 45 °C till fully dry.
12. Coated slides can be stored dry for several months at room temperature.
13. Prepare an activating antibody solution. Use an anti-CD3 antibody: either HIT3a (BD Pharmingen, #555337) or UCHT1 (BD Pharmingen, #555330), 10 μg/ml in 400 μl PBS, per chamber. This solution should be prepared shortly before slide preparation.
14. Fill each chamber with 400 μl of the activating antibody solution.
15. Incubate the slide for 2 hr at 37 °C or, preferably, overnight at 4 °C.
16. Remove the activating antibody solution and immediately wash the chamber twice with 300 μl PBS. From this point, make sure the chambers remain filled with buffer.
17. Store the PBS-filled slide at 4 °C. We suggest using the prepared slide chambers within 1 week.

3. T-cell Activation and Imaging

1. Prepare imaging buffer [1.25 ml HEPES (1 M), 10 ml FCS, 38.75 ml RPMI without phenol red]. Imaging buffer can be stored at 4 °C after filtration.
2. Activate the Zeiss LSM 510 Meta microscope. Warm the microscope heatable mounting frame to 37 °C. Select “Expert mode”.
3. Click the “Acquire” tab and open the following tabs: Lasers; Microscopy; Configuration; Scan; Time Series.
4. Activate the needed lasers. For excitation of mCFP and mYFP, set the Argon/2 laser to “standby” for 5 min, then to “on”.
5. If you have performed this live-cell imaging assay before, using the same fluorophores, open the resulting LSM file, click the “reuse” icon, and continue from step 9. Otherwise continue from step 6.
6. Define channels using filters appropriate for the excitation and emission of the fluorescent proteins used.
7. In the “Scan control” window, select the Z stack option.
8. Enter the following scan parameters: Number of slices: 5; Interval: 0.5 μm; Current slice: 3.
9. Prepare an Accublock Digital Dry Bath (Labnet) at 37 °C near the microscope.
10. Warm the imaging buffer to 37 °C.
11. Prepare the stored slide by aspirating the PBS, replacing it with 600 μl of warm imaging buffer.
12. Incubate the slide at 37 °C. Keep the slide in the incubator until step 13, for at least 15 min.
13. Collect 5 × 10⁵ transfected T cells.
14. Centrifuge cells for 2 min at 400 × g.
15. Discard the supernatant.
16. Resuspend cells in 1 ml warm imaging buffer, and transfer them to a 1.5-ml Eppendorf tube.
17. Place the test tube on the Accublock Digital Dry Bath.
18. Take the warmed slide from the incubator, and mount it on the warmed microscope mounting frame.
19. Click the “VIS” icon.
20. Mix the cell suspension and remove 1-2 μl.
21. Insert the tip of the pipette into the imaging buffer above the bottom of the slide. Avoid scratching the slide.
22. Seed the cells over the objective’s aperture.
23. Immediately begin visually tracking the fluorescent cells as they descend. Select a cell that displays high fluorescence in both channels.
24. Before the cell reaches the surface of the slide, activate the LSM mode.
25. Toggle only one excitation laser. Select a laser for which you have a DIC channel.
26. Set Zoom to 1. Click the “Fast XY” icon. Click the “Stop” icon. Using the Crop tool, select an ROI centered on the cell.
27. Set Zoom to 3. Click the “Fast XY” icon, then manually set the focus to optimally view the cell. Click the “Stop” icon.
28. Toggle all the needed excitation lasers.
29. Begin recording by clicking the “StartT” icon. Image the cell for the entirety of the spreading process (commonly 5 min). When finished, click the “Stop” icon.
30. Save the file.
31. To acquire additional cells, click the “VIS” icon. If there are cells that did not yet come into contact with the slide’s bottom, continue from step 23. Avoid imaging cells that have already begun the spreading process. Otherwise proceed to step 32.
32. If the observed slide area is mostly devoid of cells, add another aliquot of cells (continue from step 20). Otherwise proceed to step 33.
33. To acquire additional cells, move the slide so that the area above the objective’s aperture is devoid of cells.
34. Add another aliquot of cells (and continue from step 20).
35. After performing all the desired imaging, open a saved LSM file.
36. Click “Gallery”, “Time+Z” and “Subset”.
37. Select the relevant time range and Z stack.
38. Save the new file.
39. Perform steps 35-38 for all the imaging files.

4. Imaging Data Analysis

1. Open the LSM files using Imaris software (Bitplane AG, Zurich Switzerland).
2. Click “Surpass”. Click the “Image Processing” menu, and select “Swap Time and Z”. This will assist in the correct cropping of the image.
3. Click the “Edit” tool bar and select the “Crop 3D” option in the drop menu. Crop the image to include only the area surrounding the cell. We suggest cropping a 300 × 300 pixel square centered on the cell. Note that the shape and location of the cell can change over time. Observing the t axis, make sure that the cell is within the cropped region at all time points.
4. Click the “Image Processing” menu, and select “Swap Time and Z” to restore the original temporal separation of the image.
5. Press the “Coloc” button on the main tool bar. Select “Build Time Dep. Coloc”. Imaris will automatically calculate intensity thresholds for each channel at each time point.
6. View the results of the colocalization analysis by selecting “Channel Statistics”. Export the data by clicking “Export”.
7. Repeat steps 1 to 6 for each imaged cell. We recommend that at least 50 cells be analyzed in this manner.
8. Calculate the mean Pearson’s colocalization coefficient and its standard error or deviation.