After staining, cells were analyzed using a Cytomics FC 500 or CyAn ADP. Isotype matched rat anti mouse IgG antibodies served as a negative control. Cytokine done assay Inhibitors,Modulators,Libraries and ELISA To prepare for cytokine assay, plasma samples were col lected from all subjects before and after re ceiving Stem Cell Educator therapy, and kept at 80 C in a refrigerator. To determine cytokine levels, human plasma samples were quantified using commercial ELISA kits following the manufacturers Inhibitors,Modulators,Libraries instructions. We purchased human IL 1, IL 6, IL 10, TNF and TGF B1 ELISA kits from Biolegend, Inc. Western blot CB SCs were collected and solubilized with Complete Lysis M buffer with a cocktail of protease inhibitors. Cell samples were mixed with a loading buffer, 2% SDS, 10% gly cerol, 50 mM dithiothreitol, 2 mg of bromphenol blue in a volume ratio of 1 1, boiled, loaded and sepa rated by electrophoresis on 10% SDS gel.

The separated proteins were then transferred to a nitrocellulose membrane, Inhibitors,Modulators,Libraries blocked with 5% non fat dry milk in Tris buffered saline with Tween for one hour and incubated with different anti bodies including rabbit anti human cellular inhibitor of apoptosis protein 1 and cIAP2 monoclonal an tibodies and mouse anti human TNF RI or TNF RII monoclonal antibodies at 1 1,000 dilution, diluted in PBST for two hours at room tempe rature. After washing, the blot was exposed to a horserad ish peroxidase conjugated secondary antibody in PBS T. The immunocomplexes were visualized by the enhanced chemiluminescence method. Beta actin served as an internal loading control.

TNF treatment and cell proliferation To determine the effects of TNF on the proliferation of CB SCs, CB SCs were treated with recombinant human TNF at different doses, such as 100, 50, 25, 12. 5 and 0 ngml, in non tissue culture treated 24 well plates at 37 C, 8% CO2 conditions. After three days, cell proliferation was evaluated Inhibitors,Modulators,Libraries using a CyQUANTR Cell Proliferation Assay Kit. Cell fluorescence was mea sured using a Synergy HT Multi Detection microplate reader equipped with filters for 480 nm excitation and 520 nm emission. The optical values were analyzed using the manufacturers software KC4 v3. 1. Cell sorting and co cultures To purify CD14 monocytes, the freshly isolated periph eral blood mononuclear cells were initially in cubated with 2.

5% horse serum to block Fc receptor binding and then incubated with FITC conjugated CD14 antibody for 45 minutes at 4 C and subjected to cell sorting using MoFlo. After confirming the purity of the population, CD14 monocytes were collected and used in different in vitro co culture experiments with CB SCs. Culture of CB SCs were performed as pre viously described. Purified CD14 Inhibitors,Modulators,Libraries monocytes were co cultured with CB SCs at a ratio of 1 5 of CB SCs mo nocytes. After co culture with CB SCs for 18 hours, floa ted cells Tipifarnib myeloid were collected for apoptotic assay by flow cytometry.