Fluorescence microscopy of stimulated Melan-A–specific CTLs demonstrate colocalization of exposed PS with lipid rafts. (A) Exposed PS is distributed nonhomogenously in patches. Melan-A–specific CTLs were stimulated for 4 hours with anti-CD3/CD28–coated beads (middle image) and isolated via magnetic depletion of the beads. Activated CTLs were stained with annV-FITC for 30 minutes at RT. CTLs were fixed with 2% PFA in annV-binding buffer and transferred onto poly-l-lysin–coated slides. Control CTLs were treated equally without stimulation (left image). Anti-fas mAb (clone CH-11) treated apoptotic/necrotic annV^high CTLs are shown on the right image. (B) To detect colocalization of PS with membrane lipid rafts, CTLs were labeled with CTx-B and an anti–CTx-B–Alexa594-conjugated rabbit serum prior to annV-FITC staining. CTLs were fixed with 2% PFA in annV-binding buffer and transferred to poly-l-lysin–coated slides. Transmission light and emitted fluorescence was detected with a Zeiss Axioscope2plus microscope. An overlay of green and red fluorescence (yellow) exhibits a colocalization of PS with raft domains. (C) Melan-A–specific CTLs were preincubated with anti–MHC-I (blue) and CTx-B (lipid rafts, red) and stimulated with Melan-A–loaded T2 cells for 2 hours. AnnV-FITC (green) staining was performed and PS exposure at the immunologic synapse was visualized by overlay of fluorescence emission (yellow).