Wednesday, October 29, 2003
Description A method to measure Ag-specific Calicium flux in mice cells. After immunization of mice lymphocytes are collected and restimulated in vitro. In parallel, antigen-presenting cells are loaded with the studied Ag. Cells are mixed during the experiment to measure the resulting Calcium flux. Procedure Mice are immunized s.c. with Ag/CFA. After one week draining LNC are collected and restimulated in vitro with the Ag for an additional week. In parallel, antigen-presenting cells (APC) are prepared 48h before the assay is performed by incubating splenic leukocytes from nave mice with Ag, non specific Ag or PBS as control. Loading/staining with FURA-2 AM: * Wash cells with RPMI (x2). * Adjust concentration of LNC to 106/ml in RPMI. * While vortexing, add Fura-2 (solution=100X after reconstitution with 99.8 l of DMSO/vial), then add F-127 (pluronic , detergent, 1 l/106 cells). Vortex for 1 minute. * Incubate for 1 hour, culture incubator (37 C, CO2) with loose cap * After this hour wash (x3, HBSS) LNC (loaded) and APCs, turn on waterbath and calcium machine 15 mn before reading time * Mix LNC and APCs for the last wash * Resuspend at 10^6/ml in HBSS (Ca2+ free) for reading (with or without EGTA, depends on the cells) Recipes Supplies Tips Intracellular calcium was measured quantitatively on an F-2500 spectrofluorometer (Hitachi Instruments Inc., Naperville, IL). Excitation wavelengths were of 340 and 380 nm, and fluorescence emission was measured at 510 nm. Ca2+ (CaCl2 2 mM final) was added after 60s in the medium, giving the begining of the signal. (责任编辑:泉水) |