Effect of metformin on 24 hour culture of SLE CD4 T cells and Healthy Control CD4 T cells

Investigate the effect of 1.0 mM Metformin on CD4 T cells from Systemic Lupus Patients and compare to Healthy Control CD4 T cells Peripheral blood CD4+ T cells from 5 female Systemic Lupus patients of varying disease activity, and 5 healthy controls were cultured in cRPMI. CD4 T cells were isolated by Rosette Sep (Stem Cell) antibodies and ficoll-paque centrifugation. These antibodies deplete CD8, CD16, CD19, CD36, CD56, CD66b, gamma/delta TCRs. Cells were cultured in 24 well plates for 24 hours in paired conditions for each donor: plate bound CD3 antibody and soluble CD28 for stimulated in complete RPMI media, each donor treated with 1.0mM Metformin or untreated. After culture. cells were lysed in buffer RLT (Qiagen) with 0.1% 2-mercaptoethanol and stored at -80C until further isolation with Qiagen RNeasy mini kit. RNA was quantified with a Biotek Epoch spectrophotometer and 200 ng of each sample was used for fragmenting and labeling synthesized dsCDNA according to Thermo Fisher instructions for HTA 2.0 microarrays. Scanning was performed with GeneChip Scanner 3000 7G, washing and staining with the GeneChip Fluidics Station 450.