Assessing T-cell Receptor-Induced Calcium Influx Using a Calcium Indicator Dye

To begin, prepare a 1 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 at a concentration of 6 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 1-to-1 ratio to get 5 to 6 million cells per milliliter. Next, to a 12-well tissue culture plate, add 1 milliliter of cell suspension per well, per experimental condition.

Prepare a single-stained control sample for calcium-free Indo-1 AM by adding EGTA to Indo-1 dye-loaded cells. Repair 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 2 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 1 milliliter of pre-warmed cRPMI, pellet again, and decant the supernatant.

Resuspend the cells in 1 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 1 milliliter of the cell volume.

After rest, pellet the cells, and wash the pellet by resuspending in 1 milliliter of cRPMI chilled to 4 degrees Celsius. Repeat this step, and place the tubes on ice. Resuspend individual samples in 500 microliters of phenol red-free cRPMI, using a 5-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 conical tube in half, and discard the top of the tube. Fill 3/4 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 Plus 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 or 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 3,000 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.

Unmix the single-stained controls by clicking on the Unmix button. Once unmixing is complete, create sequential plots using the Unmixed Worksheet, such as SSCA versus FSCA for removing debris from the sample, and SSCA versus SSCH 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 deionized water for 2 to 3 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 SSCA versus SSCH. Complete defining the axis parameters by left-clicking on the axis. Create a plot with SSCA versus viability dye signal. Gate the live cells, which are negative or 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 one 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 3,000 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.