FACS and TLC of GM3 expression in p PBMCs. PBMCs were either resting or stimulated with PHA or PHA/IL-2 and analyzed for total and surface expressed GM3. (A,B) TLC of total GSLs extracted from control PBMCs (C; p3: lanes 3, 5, and 7) and p PBMCs (p3: lanes 2, 4, and 6). Lane 1: GSL standards. Lanes 2 and 3: Resting PBMCs. Lanes 4 and 5: PHA-activated PBMCs. Lanes 6 and 7: PHA/IL-2-activated PBMCs. (A) TLC of total GSLs. (B) TLC overlay to confirm the position of GM3. (C) Band intensity of GSLs represented on the TLC plate in panel B was measured by ImageJ software, compensated to background levels and fold difference in p-PBMC expression levels calculated based on control values. GlcC indicates glucosylceramide; GalC, galactosylceramide; LC, lactosylceramide; P^k, globotriosylceramide; P, globoside or globotetraosylceramide; Gb₅, globopentaosylceramide; GM3, ganglioside. (D) Histogram plots representing percentage of PBMCs labeled with anti-GM3 GAM-APC were analyzed, and background compensated to isotype controls. (Top panel) Control PBMCs (C-p3). (Bottom panel) p PBMCs (p3). (Left) Resting PBMCs. (Center) PHA-activated PBMCs. (Right) PHA/IL-2-activated PBMCs. (E) MFI of surface expressed GM3 was measured and fold difference calculated based on control values.