Steroid basmak

Intracellular staining and a flow cytometry assay were performed, as previously described [ 35 ]. Single-cell suspensions from the spleens of model mice at 4 weeks were prepared. Dispase was added as a stimulus at U/ml; as a positive control, 2 μg/ml PHA (Fluka; Sigma, Louis, MO) were added in the absence of dispase. As a negative control, PBS was added in the absence of dispase. After 1 h of incubation, Brefeldin A (Sigma) at a final concentration of 10 μg/ml was added to cultures to enable intracellular protein to accumulate in all stimulations. After incubation for a total of 5 h, the cells were harvested, washed twice with PBS, fixed with 4% paraformaldehyde, and permeabilized with PBS containing % saponin (Fluka) plus % BSA buffer overnight at 4 °C. They were then stained with anti-CD4, anti-CD8, anti-IFN-γ, or isotype control Abs (BD) for 30 min at 4 °C and washed twice with PBS before being resuspended in PBS containing % BSA and % NaN 3 . Cells were acquired using a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA), and FACS data were analyzed using FlowJo (BD Biosciences). Isotype-matched controls for cytokines were included in each staining.

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Steroid basmak

steroid basmak

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