Monitoring B Cell Activation with Antigenic Liposomes using a Calcium-Flux Assay
Trascrizione
To monitor B cell response to antigenic liposome activation, resuspend 1.5 x 107 splenocytes in calcium flux loading buffer, and add 1.5 micromolar Indo-1 to the cells, inverting the solution in the tube several times to mix. After a 30-minute water bath incubation at 37 degrees Celsius protected from light, add 5 times the volume of calcium flux loading buffer, and centrifuge the Indo-1-labeled cells.
For B cell gating, stain cells with anti-CD5 and anti-B220 antibodies in 0.5 milliliters of loading buffer at 4 degrees Celsius for 20 minutes protected from light. At the end of the incubation, wash the cells in fresh calcium flux loading buffer, and resuspend the pellet at 1 to 2 x 107 cells per milliliter of fresh calcium flux running buffer for storage on ice protected from light, until analysis.
Next, add 0.5 milliliters of cells to a capped 5-milliliter round-bottom polystyrene tube, and warm the cells to 37 degrees Celsius in a water bath. After 3 to 5 minutes, transfer the tube to a 37 degrees Celsius water-jacketed chamber connected to a recirculating water bath, and run the tube in the water jacket on the flow cytometer.
After collecting 5,000 to 10,000 events per second, allow the cells to stabilize for 15 to 30 seconds, and re-initiate the data acquisition, collecting the data for at least 10 seconds to establish a background reading. At the 10-second mark, quickly remove the tube from the flow cytometer, and add the appropriate experimental concentration of antigenic liposomes. Then, pulse vortex the cells, and read the tube on the cytometer for an additional 3 to 5 minutes.
