Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Mice were maintained at the University of Colorado Anschutz Medical campus in accordance with IACUC protocols. All mice were euthanized according to AAALAC standards.

1. Preparation of immune cells from the mouse spleen

NOTE: Euthanize naïve mice with CO₂ euthanasia. C57BL/6 mice purchased from Jackson Laboratories and bred in-house are used for experiments at 6-12 weeks of age. Both male and female mice are utilized for experiments.

1. Disinfect the mouse skin with 70% ethanol in order to reduce the possibility of external contaminants from getting into the sample.
2. Dissect the mouse spleen using dissection tools, surgical scissors, and forceps. Harvest the spleen and place the detached spleen into 50 mL conical tube with ~5 mL of complete RPMI media (cRPMI) on ice.
   NOTE: Complete RPMI is comprised of 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin.
   1. To harvest the mouse spleen, make a 5 cm incision into the fur and skin along the left side of the mouse halfway between the front and back legs with scissors. Open the body cavity ~5 cm and remove the spleen using forceps. The spleen is the color of a kidney bean and is longer and flatter than the neighboring kidney.
3. Pour the contents of step 1.2 onto a sterile 70 µm filter attached to a new 50 mL conical tube. Mechanically separate the spleen into a single cell suspension by pushing the spleen through the filter with a 5 mL syringe plunger until no spleen pulp remains on the filter surface.
4. Pellet the splenocytes utilizing a swing-out rotor countertop centrifuge at 500 x g for 5 min at 4 ˚C.
5. Carefully decant the supernatant. The pelleted splenocytes remain at the bottom of the 50 mL conical tube.
6. To remove red blood cells, re-suspend the pelleted splenocytes in 1 mL of ACK lysis buffer for 3 min according to the manufacturer's instructions. Mix well.
7. Quench the ACK lysis buffer with 15 mL of cRPMI.
8. Pellet the lymphocytes as directed in step 1.4.
9. Re-suspend the cell suspension in 5 mL of cRPMI in a 50 mL conical tube. Place the samples on ice.
   1. To count the cells, transfer 10 µL of the cell suspension to a 0.6 mL microcentrifuge tube and dilute at a 1:1 ratio with Trypan blue solution. Then, transfer to a hemocytometer or automated cell counting system.
10. After counting, depending on the cell concentration in cell suspension as in step 1.9, pellet the cells in a tabletop centrifuge at 500 x g at 4 ˚C for 5 min to prepare for the addition of cRMPI media containing Indo-1 AM ester dye.
11. Calculate the volume for re-suspension of the cells to achieve 10-12 x 10⁶ cells/mL. This is a 2x concentration of the total cell count required.
12. Re-suspend the cells in the volume of cRPMI calculated in step 1.11. Place the cells at 37 ˚C with 5% CO₂ in either a 50 mL conical tube with the lid vented or a tissue culture plate.

2. Indo-1 ratiometric dye and fluorescent antibody labeling

1. As per the manufacturer's instructions, add 50 µL of DMSO into a vial containing 50 µg of Indo-1 AM. The concentration of the stock solution is 1 µg/µL.
   NOTE: DMSO is provided in micro-vials with the kit to prevent any oxidation of DMSO.
2. Dilute the stock aliquot (1 µg/µL) into the appropriate experimental volume (established in 1.11) of cRPMI at a concentration of 6 µg/mL, a 2x concentration. Do not store Indo-1 in an aqueous solution.
   NOTE: Other buffers with added calcium can be used depending on the cell needs.
3. Set complete media with Indo-1 aside in a water bath at 37 ˚C.
   NOTE: Use the minimum concentration of Indo-1 AM ester necessary to obtain an adequate signal. This needs to be titrated for varying cell types. Typically, a concentration between 3-5 µM is sufficient. For primary T-cells, loading cells with Indo-1 at a concentration >5 µg/mL increases mean fluorescent intensity (MFI) above a log of 6 on spectral flow cytometers. If this occurs, decrease UV laser intensity and titrate the Indo-1 to a lower concentration.
4. Dilute previously prepared cell suspension (in step 1.12), 1:1 in cRPMI, including the 2x Indo-1 media made in step 2.2. Ensure that the cells are at a concentration between 5-6 x 10⁶/mL.
   NOTE: Do not dye load unstained single stain control or surface marker single stain cell controls with Indo-1. Adding Indo-1 to single stain antibody controls will create a multi-color sample due to the Indo-1 added to single stained cells. Include indo-1 (calcium-free) EGTA added control to the sample plate created in step 2.6.
5. Add 1 mL of cells/well/experimental condition into a 12-well tissue culture plate.
6. Create a single stained control for Indo-1 (calcium-free) sample using previously Indo-1 dye-loaded cells and add EGTA to a final concentration of 2 mM. EGTA will chelate calcium in the cell suspension, giving a cleaner control sample.
   1. Create an Indo-1 positive control (Indo-1 calcium-bound) by adding 50 µM of ionomycin to the dye loaded cell suspension at the flow cytometer. Ionomycin is a membrane permeable calcium ionophore that binds calcium ions and facilitates the transfer of calcium ions into cells.
7. Create a well for biological negative control with no fluorophores or Indo-1 dye.
8. Create a well for single stain controls for any additional cell populations of interest (antibodies user defined) using antibody conjugated fluorophores in a flow cytometry panel design. These will not include the Indo-1 ratiometric dye.
9. Add the Fc receptor blocking antibody (~2 µg/1 x 10⁶ cells), as per the manufacturer's instructions, in advance of adding additional fluorochrome-conjugated antibodies for surface staining.
10. Incubate the plate for 45 min at 37 ˚C with 5% CO_2.
11. Resuspend the cells in a 12-well plate by gently tapping the plate every 15 min.
12. Mix and remove the entire volume of cells suspended in the media from the 12-well plate. Then add the cell suspension to 1.7 mL microcentrifuge tubes.
13. Pellet the cells in a microcentrifuge at 500 x g for 5 min at room temperature. Decant the supernatant and wash the cells by adding 1 mL of pre-warmed cRPMI. Pellet again and decant the supernatant.
    NOTE: Washing the cells removes any remaining FC blocking antibody.
14. Resuspend the cells in 1 mL of cRPMI in 1.7 mL microcentrifuge tubes and incubate each tube at 37 ˚C for an additional 30 min at 5% CO₂, leaving the top of the tube slightly open to allow for gas exchange. This allows complete de-esterification of the intracellular AM esters.
15. Transfer the cells back into a 12-well tissue culture plate, if needed. Additional user defined titrated fluorochrome-conjugated antibodies can be added at a resting phase.
    NOTE: Markers used in the methods panel include: Ghost 540, CD4, CD8, TCRβ, TCRγδ, CD25, and CD1d/α-galcer tetramer. Markers are chosen to analyze T-cell subsets.
    1. Create a master mix of all the user-defined fluorochrome-conjugated antibodies according to the cell types of interest at a previously titrated volume into a 1.7 mL microcentrifuge tube.
    2. Add a calculated master mix for 1 mL of the cell volume, dependent on titrated antibodies to resting cells.
       NOTE: The advantage of staining at step 2.15 instead of the later step 2.18 is that staining at step 2.15 will decrease the overall incubation time for the experiment. However, staining in a total volume of 1 mL at step 2.15 increases antibody usage.
16. After rest, pellet the cells at 500 x g for 5 min at room temperature.
    NOTE: Cells in a 12-well tissue culture plate can be pelleted in the plate with a plate centrifuge accessory. Otherwise, pellet the cells in a 1.7 mL microcentrifuge tube.
17. Wash the cells by resuspending the pellet in 1 mL of 4 ˚C cold cRPMI and pellet the cells again as in step 2.16. Place the cells on ice.
    NOTE: If surface staining cells separately, post de-esterification at step 2.18, less antibody volume is needed. Surface staining at this step can be performed after the rest phase in cold flow buffer (1x DPBS with added 1% FBS), using user-defined antibodies, on ice for 30 min. Wash the cells after the stain in cRPMI as in step 2.17.
    1. Add viability stain Ghost540 diluted 1:7,500 in DPBS to samples according to the manufacturer's instructions. Heat kill the cells at 55 ˚C on a warming block for single stained live/dead control.
       NOTE: Viability functional dye staining to eliminate dead cells in flow cytometric analysis is performed without FBS. FBS can interact with amine dyes as per manufacturer's instructions.
18. Re-suspend individual samples in 500 µL of cRPMI (phenol red-free) using a 5 mL polystyrene flow tube with a cap.
    NOTE: Cells can be left at 4 ˚C for up to an hour.
19. Warm every 5 mL tube containing cells, individually, to 37 ˚C using a bead bath for 7 min prior to the analysis on the flow cytometer keeping time consistent between the samples. Use a timer.

3. Bead bath tube: maintaining temperature during calcium flux analysis

1. Add bath beads to a small water bath, with no water added; warm up to 37 ˚C.
2. Carefully cut a 50 mL conical tube in half, at the 25 mL mark, with a razor blade; discard the top of the tube.
3. Fill the halved tube ¾ of the way with bath beads.
4. Place the tube created in step 3.2 into the bead bath created in step 3.1. Bring the tube and beads to 37 ˚C.
5. Plug the bead bath into an electrical outlet next to the flow cytometer in order to maintain the temperature in preparation for sample analysis.
6. Add a 50 mL conical Styrofoam rack cut to hold one tube to position the tube in place while running on the flow cytometer. This will eliminate the need to manually hold the tube for the duration of the curve analysis.

4. Acquisition of calcium influx using flow cytometric analysis

1. Log into the flow cytometer software on a flow analyzer.
2. Click on the Library tab to add Indo-1 (calcium-bound) and Indo-1 (calcium-free) fluorescent tags. Select Fluorescent Tags on the software menu on the left. Select UV laser under fluorescent tag groups and click on +add to add a fluorescent tag name (Indo-1 (calcium-bound)/Indo-1 (calcium-free)) to the library. Choose the laser excitation wavelength and the emission wavelength, and then click on Save. Continue to the experimental setup.
   NOTE: Indo-1 is excited by the UV laser at a wavelength of 355 nm. The emission wavelength of Indo-1 (calcium-bound) is 372 nm. The emission wavelength of Indo-1 (calcium-free) is 514 nm.
3. Click on the Acquisition tab. Open/create a new experiment and add the desired fluorescent tags to the experiment.
4. Create Reference Group and a new sample group/s for the experiment. Label both the samples and the reference groups as necessary.
5. Under the Acquisitions tab, set the events to record to the maximum: 10,000,000.
6. Set the stopping volume to the maximum volume: 3,000 µL.
7. Set the stopping time to the maximum time: 36,000 s.
8. Acquire single stained controls for defined conjugated-antibodies included in the multicolor panel.
9. Acquire single stained controls for Indo-1 functional dye.
   1. Add 50 µM of ionomycin to the calcium bound Indo-1 positive control. Vortex to mix. Add the flow tube to the sample injection port and click on Record.
   2. Add calcium free Indo-1 negative control to the flow cytometer and click on Record. EGTA was added in step 2.6.
   3. Unmix the single stained controls by clicking on the Unmix button.
      NOTE: All the single stained controls from all the conjugated antibodies and functional dyes must be recorded prior to unmixing the samples. For the Unmix button to function, all the reference controls need to be recorded first. Unmixing can be done before or after sample collection.
10. Once unmixing is complete, create sequential plots using the unmixed worksheet. SSC-A versus FSC-A removing debris from sample, SSC-A versus SSC-H to remove doublets. This is followed by a viability clean-up gate along with further gating on populations of interest. To create gates, click on Plot. Add a plot to the worksheet, and double click inside the gate to create a downstream gated population. Continue until all the populations of interest are accounted for.
11. Warm single stained control samples (from steps 2.6-2.9 in section Indo-1 ratiometric dye and fluorescent antibody labeling) to 37 ˚C for sequential analysis. Set up the flow cytometry software.
12. Run DI water on the cytometer for 2-3 min to ensure the fluidic stability of the flow cytometer.
13. Set up sequential plots on the unmixed worksheet by creating gates using the Polygon, Rectangle or Ellipse gate buttons. Gate to include the population of interest (i.e., lymphocytes using SSC-A vs. FSC-A [size/lymphocytes discrimination]). Negative populations appear clustered around zero whereas positive populations can be visualized by increasing MFI. Populations of interest will be user defined based on the biological question.
14. Double click inside the gate created and change Y-axis and X-axis parameters to SSC-A versus SSC-H (singlet discrimination). Complete defining the axis parameters by left clicking on the words on the axis.
15. Create a plot with SSC-A versus viability dye signal (viability gate parameters). Gate on live cells, which are negative for amine dye staining. Live cells, negative for live dead staining, will cluster at the 0 mark on both the Y and X axes.
16. Double click on the inside plot to create a new plot containing only single cell live lymphocytes. Change the Y-axis to Indo-1 (bound-calcium) (V1) and/or Indo-1 (free-calcium) (V7) versus time on the X-axis for visualization of the calcium influx.
17. Place the warmed biological sample onto the machine SIT and run the samples at 2,500-3,000 events/s on medium to allow visualization of calcium influx in limited cell number populations (e.g., iNKT).
    NOTE: By resuspending the cells at a concentration of 1 x 10⁶/mL, the cell events collected at medium flow rate should equal 2,500-3,000 events/s. Lower the flow rate under acquisition control by clicking on the drop-down menu or dilute the sample if the cell suspension has an increased concentration. Running the samples at a high flow rate can increase the spreading of the signal from one fluorophore to other fluorophores in the panel.
18. Add the next tube to the bead bath to warm to 37 ˚C.
19. In the Acquisition Control, press Record to record the initial data for 30 s to obtain a basal level of calcium signaling in the sample.
20. In the Acquisition Control, click on Stop and remove the tube from Sample Injection Port (SIP).
21. Add 30 µg of unconjugated anti-CD3 directly into the sample tube to initiate calcium influx. The final concentration per tube is 60 µg/mL.
    NOTE: There is no washing step after the addition of anti-CD3.
22. Place the tube back on machine SIP as quickly as possible and press Record in the software.
23. Record data for 7 min, watching the time elapsed under the acquisition controls in the software, to observe the time-course of calcium influx within the sample populations.
24. After 7 min, press Stop in the software and add 1 µg/mL of ionomycin. Record for 30 s to obtain the maximum calcium signal in the cells of interest. To limit carryover of ionomycin into the next sample, complete two SIT flushes on the instrument between samples.
25. Continue to the next sample and repeat the workflow until all the samples have been collected.
26. Save the experiment and click on the export zip file. Unmixed files (.fsc) are uploaded to external flow cytometry analysis software.

5. Calcium curve analysis

1. Using an analysis software⁶ for flow cytometry, convert Indo-1 fluorescent signals to a ratio for time-lapse calcium measurements. Derive parameters under the tools tab by inserting references Indo-1 (calcium-bound)/Indo-1 (free-calcium) using a linear scale. Set the minimum to zero and the maximum according to the influx of calcium ratio, typically around 10. The maximum ratio varies by cell type and MFI of Indo-1.
2. Highlight the selected parameter. Under tools, add kinetic analysis to choose the parameter by clicking on the Kinetics tab. Set the Y-axis to derived.
3. Select MFI (mean fluorescent intensity) within the Kinetics tab.
4. Add Gaussian smoothing, if desired. To add Gaussian smoothing, select the option in the pull-down statistics menu in the flow analysis software. Gaussian smoothing can be added to smooth the visualization of the calcium influx curve of analysis.
5. Create multiple ranges for statistics along the calcium flux time course for biological comparisons within the samples.
6. Drag the samples into the layout editor and add the statistics as desired. Statistical analysis varies depending on the biological question. For publication purposes, multiple replicates are analyzed using t-test or ANOVA.
7. For a moment of time analysis, set the gate on a specific moment in time along the calcium flux. Examine the desired surface markers and gate on the population of interest; then, assess a two-dimensional dot-plot of Indo-1 (calcium-free) versus Indo-1 (calcium bound) for gated cells within that moment in the time region.