Journal: The American Journal of Pathology
Article Title: Neural Stem/Progenitor Cells Express Costimulatory Molecules That Are Differentially Regulated by Inflammatory and Apoptotic Stimuli
Figure Lengend Snippet: Stress-induced apoptosis differentially increases CD80 expression by NSCs and CD80 ligation increases apoptosis. A: Stress was induced by culturing NSCs with hydrogen peroxide at 100 ÃÂ¼mol/L concentration for a 24 to 48 hour period ( left histograms ) or by media deprived of serum and growth factors (right histograms ). Cells were detached using EDTA-Versene, were incubated for 15 minutes at 37ÃÂ°C washed and counted for flow cytometry, 7-amino actinomycin D (7-ADD) staining was used to exclude non-viable cells. B: NSCs were incubated with TSA 100 ng/ml for 18 or 24 hours and examined by two-color staining with Annexin V and 7-AAD and anti-CD80 or anti-CD86 ( left dot blot ). The expression of B7 molecules was measured by gating on early apoptotic cells (Annexin-V positive, 7AAD negative) vs. live cells (Annexin and 7-AAD negative). The green background is the isotype control whereas the red shift is the anti-CD80 or anti-CD86 antibodies. The expression of CD80 is up-regulated in cells undergoing apoptosis ( left histograms ) as compared with CD86 where a slight up-regulation occurs after a 24-hour time period ( right histograms ). C: Actively proliferating C17.2 NSCs were left untreated, incubated with anti-CD80 (50 ÃÂ¼g/ml), TSA (100 ng/ml), TSA plus anti-CD86, TSA plus anti-CD80 (5 to 10 ÃÂ¼g/ml), TSA plus CD80 F(ab), TSA plus mIg, or TSA plus rat IgG2a and the percentage of apoptotic cells measured by Annexin and 7-AAD staining. Dot blots of apoptotic cell staining after 18 hours of culture comparing various culture conditions ( left ). The early apoptotic cells are seen in the lower right quadrant . D: Mean ÃÂ± SE of percentage of apoptotic cells from triplicate cultures. Apoptotic cells were calculated as the sum of early and late apoptotic cells. These data were reproduced in three independent experiments (each done in triplicate). The effects of TSA alone is demonstrated by an increase in apoptotic cells compared to untreated cells ( P
Article Snippet: For surface expression, proliferating NSCs were detached using incubation with EDTA for 30 minutes at 37ÃÂ°C, the cells were washed, counted and resuspended in staining buffer, treated with FC block (Pharmingen), and stained with labeled antibodies.
Techniques: Expressing, Ligation, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Staining, Dot Blot