18 X{5vX
T\/y
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number Ag 9
vU7
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The l4F%VR4KT
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at !%CWZZ 6u
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The s'!Cp=xQF"
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 S0z
D"T
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, ZGexdc%
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive ?qPo=~y01
control for Ca2+ induction. The data thus obtained was analyzed with the software Win MqW7cjg
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on azCf
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 "|EM;o
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were JZ)RGSG i
harvested and fractionated as detailed above, and the S-100 fractions were assessed by |n2qVR,
immunoblotting for presence or absence of annexin I. -.1y(k^4E
Mouse bone marrow transduction and transplantation. Bal$+S
Retrovirus-mediated transduction of mouse bone marrow cells was done by published J?V? R
methods49. Prestimulated low-density bone marrow cells were infected with high-titer r(:5kC8K
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated A
9( x
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing * aN
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by YN/|$sMD|
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) ?M8dP%&r
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone &TUWW