18 OcyiL)tv 5
Jurkat cells cultured in calcium-free RPMI-1640 medium (Gibco BRL; number d"QM;9
22300-107) containing calcium-free 10% FBS were triggered by anti-Fas IgM. The 5e~ j
treated cells were harvested at the indicated time points and incubated with Fluo-3-AM at Y#Sd2h,^X
a final concentration of 1 micromolar for 30 min at 37°C (Scoltock et al., 2000). The 5#N"WHz!
labeled cells (50,000 cells per treatment) were then analyzed by exciting the cells at 488 tIc0S!H#
nm and examining the fluorescence emission of Fluo 3 at 530 nm with a FACS Scan, P':]A{<Z
(Becton Dickinson). A one micromolar concentration of LPA was used as a positive ;p?42rCIcl
control for Ca2+ induction. The data thus obtained was analyzed with the software Win %B*<BgJ;4F
MDI 2.8 and represented as contour plots.The effect of chelating intracellular calcium on ySPlyhGF
translocation of annexin I was studied by culturing Jurkat cells in the presence of 10 }R?v"6aBS
micromolar BAPTA-AM, with or without the addition of anti-Fas IgM. Cells were wz|Q%.%?[
harvested and fractionated as detailed above, and the S-100 fractions were assessed by 9!Q
$GE?vl
immunoblotting for presence or absence of annexin I. dvrvpDoE.
Mouse bone marrow transduction and transplantation. ImT+8pa
Retrovirus-mediated transduction of mouse bone marrow cells was done by published TYmUPS$
methods49. Prestimulated low-density bone marrow cells were infected with high-titer P^uP$D
retrovirus supernatant on fibronectin-coated plates. Retrovirus supernatant was generated :_I
wc=
in the phoenix-gp cells with a mouse stem cell virus–based retroviral vector coexpressing tXb7~aO
EGFP and HA–RhoH as described50. EGFP+ sorted cells were transplanted by y5Z<uwXc
intravenous injection into the sublethally irradiated (300 rads with a 137Cs irradiator) !`d
832
Rag2-/- recipient mice. At 9 weeks after transplantation, thymus, peripheral blood, bone (<g;-pZH%
marrow, spleen and lymph nodes from each recipient mouse were collected for analysis Pv/%s) &y&
of EGFP+ chimerism and hematopoietic lineage by flow cytometry. Expression of NXV~[
HA–RhoH and HA–RhoHF73F83 in EGFP+ sorted thymocytes of recipient mice was ~8n~4
confirmed by immunoblot analysis. H*:r>Lm=
Determination of renal morphology ??esB&