Anti mouse cd3e antibody

Spleens were filtered through 100uM filters in PBS, pelleted, and red blood cells lysed using ACK lysis buffer (Life Technologies). Splenocytes were plated at 2 ×10⁶ cells per well in 24 well dishes in RPMI 1640-Glutamax containing 25 mM HEPES (Invitrogen), 10% fetal bovine serum and 0.05μM 2-mercaptoethanol. CD4⁺ or CD8⁺ cells were purified from spleens and lymph nodes by positive selection using microbeads and magnetic columns (Miltenyi Biotech) via manufacturer’s protocol. T cells were cultured in the same media and at the same numbers as splenocytes. Stimulated wells were pre-coated with 1 μg/mL anti-mouse CD3e antibody (BD Biosciences) in PBS and rinsed with PBS before plating cells in media containing 2 μg/mL anti-mouse CD28 antibody (eBioscience). 72 hours after plating, cells were lysed in RIPA buffer. For western blotting, membranes were incubated with TSP-1 antibody clones SPM-321 and A6.1 (1:500, Santa Cruz) or β-actin antibody (1:3000, Sigma) followed by HRP labeled secondary antibodies, and detected using ECL solution. Densitometry analysis was performed on minimally exposed blots using ImageJ software (http://rsbweb.nih.gov/ij/) to determine a TSP-1:actin ratio for each time point, and then this ratio for each time point compared to day 0 was calculated.