Monitoring B Cell Activation with Antigenic Liposomes using a Calcium-Flux Assay

Take red blood cell-depleted splenocytes in a buffer containing a cell-permeant calcium -sensitive fluorescent dye, Indo-1 AM.

Intracellular esterases cleave Indo-1 AM into membrane-impermeable Indo-1, which binds to calcium ions, changing Indo-1's fluorescence signal from blue to violet.

Introduce fluorophore-conjugated antigen-specific antibodies that bind specifically to the respective antigens on cells.

Using flow cytometry, identify fluorophore-labeled B cells and measure their Indo-1 violet-to-blue fluorescence ratio.

Now, treat the cells with antigenic liposomes comprising IgM Fab fragments conjugated to liposome bilayers.

The B cell receptors bind to the liposome-coupled antigens, activating the associated cytoplasmic tyrosine kinases and triggering phospholipase-C gamma-2 activation.

Activated phospholipase-C cleaves the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, generating second messengers, including inositol 1,4,5- trisphosphate, or IP3.

IP3 binds to IP3-gated calcium channels on the endoplasmic reticulum, releasing calcium ions into the cytoplasm.

These calcium ions bind the unbound Indo-1 molecules, increasing violet fluorescence.

A higher Indo-1 violet-to-blue fluorescence ratio in labeled B cells confirms their successful activation by antigenic liposomes.

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.