18 6iOAYA=
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number j\<S 6%p#R
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The DzMk eX
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at 4l:+>U@KU
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The >"b"K{t
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 '=xl}v
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, Z]dc%>
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive &x}JC/u]fd
control for Ca2+ induction. The data thus obtained was analyzed with the software Win 08Gr
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on ,wjL3c
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 AkF3F^
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were OpfFF;"A'
harvested and fractionated as detailed above, and the S-100 fractions were assessed by j"]%6RwM]
immunoblotting for presence or absence of annexin I. 49yN|h;c!
Mouse bone marrow transduction and transplantation. ZWv$K0agu
Retrovirus-mediated transduction of mouse bone marrow cells was done by published UA^E^$f:
methods49. Prestimulated low-density bone marrow cells were infected with high-titer
FM5$83Q
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated se-}d.PwL
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing }[|"db
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by p<