For EGFR phosphorylation analysis, cells were fixed in 4% paraformaldehyde for 15 minutes, washed with PBS, permeabilizaed with 0. 3% Triton X 100 for etc 5 minutes, washed, incubated with anti phospho EGFR or EGFR anti body for 1 h at 4 C, and then with an FITC labelled sec ondary antibody for 45 min at 4 C. After washing, the cells were analyzed with a Flow Cytometer. Data analysis was performed using WinMDI 2. 7 software. Induction of apoptosis Jurkat T cells were cultured in RPMI 1640 with 10% FBS at 37 C in 5% CO2. Apoptosis was induced in Jurkat T cells by overnight exposure to 400 uM H2O2 in serum free RPMI medium. To distinguish between cells in the early or late Inhibitors,Modulators,Libraries stages of apoptosis, staining with Annexin V FITC was combined with pro pidium iodide staining.

Afterwards, cells were immediately analyzed by flow cytometry. Cells in the early stage of apop tosis were negative for PrI but stained with Annexin V FITC, whereas in the late stage apoptotic Inhibitors,Modulators,Libraries cells stained for both PrI and Annexin V FITC. Jurkat T cells treated in this way were about 90% late stage apoptotic cells. Phagocytosis assays Phagocytosis of particles Microglial cells seeded in 96 well plates or in 25 mm2 flasks were incubated with medium, 1 ug ml of sPLA2 IIA, 100 UI ml of interferon at 37 C for 24 h, in the presence or absence of the indicated inhibitors. After Inhibitors,Modulators,Libraries 24 h, the phagocytic ability of the cells was mea sured using FITC dextran as a tracer. Briefly, cells were exposed to 0. 1 mg ml of FITC labelled dextran for 2 h.

Non internalized particles were removed by vigorously Inhibitors,Modulators,Libraries washing three times with cold PBS prior to measuring fluorescence at 480 nm excitation and 520 nm emission on Inhibitors,Modulators,Libraries either a Flow Cyt ometer or a Fluoros kan multiwell plate reader. As a background, the cultures without FITC dextran were used. Each culture condition was performed in quadru plicate, and three independent experiments were per formed. To visualize the internalized dextran, cells were also analyzed on a Leica TCS SP5X confocal microscope with a ��60 oil objective. Phagocytosis of apoptotic cells Phagocytic assays were performed on BV 2 cells after 24 h incubation in the presence of the inflam matory stimuli. Apoptotic Jurkat T cells were used as target cells. Briefly, PrI labeled apoptotic Jurkat T cells were added to the BV 2 cells at a 8 to 10,1 ratio and incubated at 37 C in 5% CO2 for 2 h in DMEM medium.

Then, BV 2 cells were washed gently with cold PF-2341066 PBS and trypsinized by incubating them with a solution 0. 25% trypsin EDTA for 5 minutes to remove uningested cells. Afterwards, cells were fixed, stained with a PE conjugated CD68 antibody and ana lyzed by flow cytometry. PE fluorescence was analyzed in FL2, while red fluorescence from PrI was analyzed in FL3. To quantify phagocytosis, PrI fluorescence was analyzed only in the cell populations exhibiting PE CD68 positive staining. The BV 2 microglia cells were positive for PrI fluorescence only if they had ingested PrI labeled Jurkat T cells.