18
rd\: . Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number
tpQ?E<O 22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The
' ]'V?@H]4 treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at
tUXly|k a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The
ob(S /t labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488
m&&Y=2 nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan,
[.[|rnil (Becton Dickinson). A one micromolar concentration of LPA was used as a positive
:@=;WB*0 control for Ca2+ induction. The data thus obtained was analyzed with the software Win
1Bl;.8he.) MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on
E{B8+T:3 translocation of annexin I was studied by culturing Jurkat cells in the presence of 10
$?FA7=_ micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were
}r _d{nhi harvested and fractionated as detailed above, and the S-100 fractions were assessed by
=o? Q0 immunoblotting for presence or absence of annexin I.
eX0ASI9 Mouse bone marrow transduction and transplantation.
.`;
bQh'! Retrovirus-mediated transduction of mouse bone marrow cells was done by published
3~5 %6 ` methods49. Prestimulated low-density bone marrow cells were infected with high-titer
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