18 qJ%AbdOI8
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number 3Ued>
8Gv
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The o8!uvl}:9
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at 3R)_'!R[B
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The b5t:">wC
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 ! w2BD^V-
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, wgFAPZr
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive >A
?{cbJ
control for Ca2+ induction. The data thus obtained was analyzed with the software Win NhF<2[mt
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on M\6u4p!G!
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 ]m@p? A$
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were 1/ <Z6 ?U
harvested and fractionated as detailed above, and the S-100 fractions were assessed by P?$Iht.^
immunoblotting for presence or absence of annexin I. O#3PUuE%d
Mouse bone marrow transduction and transplantation. ObEp0-^?
Retrovirus-mediated transduction of mouse bone marrow cells was done by published vuO~^N]G
methods49. Prestimulated low-density bone marrow cells were infected with high-titer q~G@S2=}0}
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated &hSF
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing :!<