5% Tween 20 in PBS followed by another resuspension in washing Crizotinib cost buf fer. The labeled cells were detected Inhibitors,Modulators,Libraries in the green channel Inhibitors,Modulators,Libraries of a flow cytometer, CyAn Inhibitors,Modulators,Libraries ADP. FITC conjugated mouse mono clonal IgG1 was used as isotype control. MTT assay Cell viability was estimated by the enzymatic conversion of 3 2,5 diphenyltetrazolium bromide to formazan crystals in live cells. Formazan was dissolved in dimethyl sulfoxide at 90 min after addition of MTT to the culture med ium, and quantified by a spectrophotometer or a micro plate reader at 560 nm. Annexin V and propidium iodide binding assay The OPC cultures were maintained in 60 mm dishes and subjected to various experimental treatments. At 0, 24, and 48 h after these treatments, the culture medium containing detached dead cells was collected, and the attached cells were washed once with 2 ml of Ca2 and Mg2 free HBSS.
The attached cells were removed from the plate by exposure to 0. 5 ml of 0. 05% trypsin at 37 C for 2 min, suspended in 2 ml of GM with 625 ug ml trypsin inhibitor, and collected into a 15 ml tube together with the saved medium and the Ca2 and Mg2 free HBSS used for wash. After centrifuge at 520 xg for 10 min, the pellet was resuspended into 0. 4 ml Inhibitors,Modulators,Libraries of binding buffer D glucose. Five ul of FITC conjugated annexin V solution and propidium iodide were added into 0. 1 ml of the cell suspension. After incubation at room temperature for 15 min in the dark, 0. 3 ml of the bind ing buffer was added to the cell suspension. To deter mine the absolute number of cells Inhibitors,Modulators,Libraries in each preparation, Flow Count fluorospheres were added at a concentra tion of 19 beads ul just before flow cytometry by CyAn ADP.
Fluores cence of annexin V FITC and PI were detected in FL 1 and FL 4 channels, respectively. Gatings and data acqui sition and analysis were carried out using Summit soft ware as described previously. Cell death and loss of mitochondrial molarity calculator membrane potential assay Rat OPCs cultured in 24 well plates were treated with the GM or the GM supplemented with IFNg for 12, 18, and 24 h. Prior to collection, cells were incubated with tetramethylrhodamine ethyl ester at 37 C for 30 min. Then, culture medium containing dead cells was collected, and cells were washed once with 0. 5 ml of the Ca2 and Mg2 free HBSS. Attached cells were removed with 150 ul of 0. 05% trypsin for 1 min, suspended in 1 ml of the GM, and collected into a 15 ml tube together with the saved medium and the Ca2 and Mg2 free HBSS used for washing. After cen trifugation with 1500 rpm for 5 min, the supernatant was aspirated and the pellet was kept on ice. Pellets were resuspended with 0. 5 ml PBS containing 5 uM DAPI and 0. 1% BSA immediately prior to analysis by flow cytometry employing a Cyan ADP Flow Cytometer.