The protocol allows investigators to simultaneously measure calcium response in primary T-cells along with assessing their subset and surface phenotype. The main advantage of this technique is that it avoids the need to isolate specific subsets of cells before examining their calcium responses. To begin, prepare a one microgram per microliter stock solution of Indo-1 AM by adding 50 microliters of DMSO into a vial containing 50 micrograms of Indo-1 AM.Dilute the stock solution into an appropriate volume of cRPMI and a concentration of six micrograms per milliliter.
Place the medium in a water bath at 37 degrees Celsius. Then use this medium to dilute a previously prepared a cell suspension at a one-to-one ratio to get five to 6 million cells per milliliter. Next, to a 12-well tissue culture plate, add one milliliter of cell suspension per well per experimental condition.
Prepare a single stained control sample for calcium-free Indo-1 by adding EGTA to Indo-1 dye-loaded cells. Prepare a well for biological negative control with no fluorophores or Indo-1 dye. Prepare an Indo-1 positive control by adding ionomycin at a concentration of 50 micromoles to the dye-loaded cell suspension at the flow cytometer.
Also, prepare a well for single stain controls for additional cell populations using antibody conjugated fluorophores. Then add two micrograms of FC receptor blocking antibody per million cells and incubate the plate for 45 minutes with tapping every 15 minutes to resuspend the cells. After incubation, mix and remove the entire volume of cells from the plate and add it to 1.7 milliliter microcentrifuge tubes.
Centrifuge and decant the supernatant. Once the cells are washed with one milliliter of pre-warmed cRPMI, pellet again and decant the supernatant. Resuspend the cells in one milliliter of cRPMI and incubate each tube, leaving the top of the tube slightly open for gas exchange, allowing complete de-esterification of the intracellular AM esters.
Next, prepare a master mix of all the user-defined fluorochrome conjugated antibodies and add it to the resting cells for one milliliter of the cell volume. After rest, pellet the cells and wash the pellet by resuspending in one milliliter of cRPMI chilled to four degrees Celsius. Repeat this step and place the tubes on ice.
Resuspend individual samples in 500 microliters of phenol red-free cRPMI using a five milliliter polystyrene flow tube with a cap. To maintain temperature using calcium flux analysis, prepare a bead bath by adding bath beads to a small water bath and warm it up to 37 degrees Celsius. Carefully cut a 50 milliliter chronicle tube in half and discard the top of the tube.
Fill three-fourths of the remaining tube with bath beads and allow it to reach 37 degrees Celsius in the bead bath. Place the bead bath near a flow cytometer, along with a styrofoam rack to position the tube. Before the analysis on the flow cytometer, warm the tubes individually to 37 degrees Celsius for seven minutes.
Log in to the flow cytometer software and click on the Library tab. Then select fluorescent tags to add calcium-bound Indo-1 and calcium-free Indo-1. Then select UV laser under fluorescent tag groups and click on add to add a fluorescent tag name.
Choose the laser excitation and emission wavelength and then click Save. Continue to the experimental setup. Click on the Acquisition tab, Open/Create a new experiment and add the desired fluorescent tags to the experiment.
Create a reference group and a new sample group for the experiment and label both the groups. Under the Acquisitions tab, set the events to record to the maximum at 10 million, stopping volume to the maximum volume of 3000 microliters, and stopping time to the maximum of 36, 000 seconds. Acquire single stained controls for user-defined conjugated antibodies included in the multicolor panel and for Indo-1 functional dye.
Add 50 micromoles of ionomycin to the calcium-bound Indo-1 positive control and vortex. Add the flow tube to the sample injection board and click Record. Then add calcium-free Indo-1 negative control to the flow cytometer and click Record.
Unmixed the single stained controls by clicking on the Unmix button. Once unmixing is complete, create sequential plots using the unmixed worksheet, such as SSC-A versus FSC-A, for removing debris from the sample and SSC-A versus SSC-H to remove doublets. Then create gates by clicking 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. Next, warm the previously prepared single stained control samples to 37 degrees Celsius for sequential analysis.
Set up the flow cytometry software and run DI in ice water for two to three minutes to ensure the fluidic stability of the flow cytometer. Set up sequential plots on the unmixed worksheet by creating gates using the polygon rectangle or ellipse gate buttons to include the population of interest. Double-click inside the gate and change the y and x-axis parameters to SSC-A versus SSC-H.
Complete defining the axis parameters by left clicking on the axis. Create a plot with SSC-A versus viability dye signal. Gate the live cells which are negative for amine dye staining.
Live cells negative for live dead staining will cluster at the zero mark on both the y and x-axis. Double-click on the inside plot to create a new plot containing only single cell live lymphocytes. Change the Y-axis to Indo-1 versus time on the x-axis for visualization of the calcium influx.
Next place the warmed biological sample into the SIT of the machine. Run the samples at 2, 500 to 3000 events per second on a medium flow rate to visualize calcium influx in a limited number of cell populations. Add the next tube to the bead bath in acquisition control.
Press Record to record the initial data for 30 seconds to obtain a basal level of calcium signaling in the sample. Then click on Stop and remove the tube from sample injection port. Add 30 micrograms of unconjugated anti-CD3 directly into the sample tube to initiate calcium influx.
Quickly place the tube back on the sample injection port and press Record in the software. Record data for seven minutes, watching the time elapsed under the acquisition controls in the software. Then press Stop in the software.
Add one microgram per milliliter of ionomycin and record for 30 seconds to obtain the maximum calcium signal in the cells of interest. Continue to the next sample and repeat the workflow until all the samples have been collected. Save the experiment and click on the export zip file.
Unmixed files are uploaded to external flow cytometry analysis software. A stable consistent signal in a plot of time versus side scatter indicated fluidic stability. The lymphocyte population was gated followed by discrimination of singlets on a plot of side scatter height versus area.
Singlets were assessed for viability. Then gating on CD19-negative population was applied. These T-cell subsets were assessed using antibodies to CD4 and CD8 by visualizing calcium-bound Indo-1 fluorescence versus time.
In individual cell populations, the plot of calcium-bound Indo-1 versus time showed a baseline prior to stimulation. A peak response as intracellular calcium levels declined and the calcium response elicited by adding ionomycin. Prior to anti-CD3 antibody addition, a weak calcium-bound Indo-1 signal was detected.
During the peak response, the cells showed a strong calcium-bound Indo-1 signal and reduced calcium-free Indo-1. At five minutes post peak, the Indo-1 fluorescence nearly returned to baseline. After ionomycin addition, a robust signal of calcium-bound Indo-1 was visualized, indicating adequate dye-loading of the cells.
Analysis of the rare populations was performed by plotting calcium-bound Indo-1 versus calcium-free Indo-1 after gating on each population of interest. And analyzing the calcium response over the entire time course for each subset. Calcium flux using full spectrum profiling can lead to high parameter flow cytometric analysis in a variety of immune subsets.
High dimensional analysis for unbiased clustering of T-cell subsets can be used in combination with high parameter flow cytometry for advanced analysis. This technique is developed for the analysis of multiple immune subsets in conjunction with one another in a bulk sample.
