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.