Bio plex protm mouse cytokine 8 plex system

To assess the suppressive activity of vasculogenically conditioned peripheral blood mononuclear cells against T cells, splenic mononuclear cells (2.5 Å) were isolated from Balb/c mice, and incubated at 10⁵ cells/well on plates coated with (BIOCOAT 96-well anti-mouse CD3 T cell activation plate; BD) or without anti-mouse CD3 and filled with 200 μL RPMI 1640 (Sigma-Aldrich) containing 10% fetal bovine serum. Coated plates were also supplemented with 2 μg/mL anti-mouse CD28 (BD bioscience). After 48 hours, cytokines in culture supernatants were quantified with the Bio-Plex Pro^TM Mouse Cytokine 8-plex system (BIO-RAD). Wells were also reacted with 20 μL Cell Counting Reagent (Dojindo, Kumamoto, Japan), and the absorbance at 450 nm was measured after 2 hours. The stimulation index was calculated by dividing the absorbance from CD3/CD28 co-stimulated cells by the absorbance from unstimulated cells. Cytokines released by vasculogenically conditioned peripheral blood mononuclear cells only were also quantified in culture supernatants using the Bio-Plex Pro^TM Mouse Cytokine 8-plex system (BIO-RAD).

Splenic lymphocytes were incubated on plates coated with an anti-mouse CD3 antibody (BIOCOAT 96-well anti-mouse CD3 T cell activation plate; BD) or uncoated control plates (2.5 × 10⁵ cells/well) with 200 μl of RPMI 1640 containing 10% FBS. An anti-mouse CD28 antibody (100 ng/mL; Affymetrix) was added to each well of the CD3-coated plate. After incubation for 48 hours, 100 μl of culture supernatant containing CD3/CD28 co-stimulated lymphocytes was removed and analysed by cytokine assay (Bio-Plex Pro^TM Mouse Cytokine 8-plex system; BIO-RAD). Ten microliters of Cell Count Regent (Nacalai Tesque) was added to each remaining 100 μl of culture supernatant and the absorbances at 450 nm were measured after 2 hours. The stimulation index was calculated by dividing the absorbance of CD3/CD28 co-stimulated cells (with background adjustment) by the absorbance of unstimulated cells.