Mtt assay

CD8⁺ B3Z T cell hybridomas were stimulated by pOVA (SIINFEKL) presented on H2K^b murine MHC class I molecules, as previously reported^{48 (link),49 (link)}. The pOVA was purchased from MBL. K^b-expressing L cells, kindly provided from Nilabh Shastri (University of California, USA), were used as APCs. The T cell hybridoma (1 × 10⁵/well) was incubated for 24 h with the APCs (1 × 10⁵/well) in the presence of titrated amounts of exogenous peptide. The culture supernatants were then added to IL-2-dependent HT-2 cells, kindly provided from Eric Huseby (University of Massachusetts Medical School, USA), (4 × 10³/well) and incubated for 24 h. Cytokine production by T cells was assessed by measuring proliferation of HT-2 cells in a MTT assay (Dojindo). All experiments were repeated three times, each with similar results.

Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Dojindo, Kumamoto, Japan). HCAECs were cultured in growth medium in 96-well plates (0.5 × 10⁴ cells/well). Following each experiment, medium was replaced with fresh MV 2 basal medium and the final sample volume adjusted to 100 μL/well. Samples were subjected to MTT assay according to manufacturer instructions and measured in duplicate, using Microplate Reader (Tecan Infinite M200).
Cytotoxicity was evaluated via fluorescence to measure the activity of dead-cell protease, which is released from cells with impaired membrane integrity, using a CytoTox-Glo cytotoxicity assay (Promega, Madison, WI, USA) according to manufacturer instructions. Briefly, HCAECs (0.5 × 10⁴ cells/well) were cultured in growth medium in 96-well plates. After each experiment, medium was replaced with fresh MV 2 basal medium (100 μL/well final volume). Fluorescence measured using a Tristar multimode microplate reader (LB 941; Berthold Technologies, Oak Ridge, TN, USA), was directly proportional to the number of dead cells. Each sample was measured in duplicate.

The effect of CYM on B16 cells proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide or MTT assay (Dojindo, Japan). B16 cells (3 × 10⁴ cells/well) were seeded onto 96-well plates and cultured as described above. After 24 h of incubation, the medium was replaced with a fresh medium with or without the sample at various concentrations. MTT (5 mg/ml) was then added; the plates covered with aluminum foil and were incubated further for 28 h. Sodium dodecyl sulphate (SDS; 10%) was then added at 100 μl per well and incubated overnight at 37°C to dissolve the formazan completely. Absorbances were obtained at 570 nm using a microplate reader (Powerscan HT; Dainippon Pharmaceuticals USA Corp., NJ, USA). To correct the absorbances, blanks containing only medium MTT and SDS were used.