Cmfda

MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan) powder, CMFDA (5-chloromethylfluorescein diacetate) and CMRA (5-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) cell staining dyes, was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Anti-CD3 antibodies and anti-CD28 antibodies were purchased from Bioxcell (West Lebanon, NH, USA). Human IL-2 DuoSet^® ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA). Staphylococcal enterotoxin E (SEE) was purchased from Toxin Technology (Sarasota, FL, USA). NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit and ECL Western blotting detection reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-CD40L antibodies conjugated with APC was purchased from eBiosciences (San Diego, CA, USA). Anti-CD40L neutralizing antibodies were obtained from InvivoGen (San Diego, CA, USA). Anti-CD40L for western blot and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-p65, anti-PARP, anti-IκBα, anti-phosphorylated IκBα (S32), anti-phosphorylated ERK (T202/Y204), anti-ERK, anti-phosphorylated p38 (T180/Y182), anti-p38, anti-phosphorylated JNK (T183/Y185), anti-JNK, anti-phosphorylated c-Jun (S73) and anti-c-Jun antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

Cell viability was evaluated using the MTT assay (Roche, Basel, Switzerland; 11465007001) according to the manufacturer’s instructions. Briefly, 20 microcapsules were placed into a well of a 96-well plate containing 100 μl culture medium, and 10 μl MTT labeling reagent was added per well and incubated for 4 h. Then 100 ml solubilization solution was added to each well and incubated overnight. The formation of purple formazan crystals was measured using a microplate reader (DTX880; Beckman Coulter, Brea, CA, USA) at wavelength of 570 nm.
Cell viability was also assessed by cell staining. Live cells in microcapsules were labeled with fluorophoresrhodamine 123 (R123), 5- (and 6-) carboxy-4',5'-dimethylfluorescein diacetate (CMFDA,C7025, Eugene, OR, USA), and dead cells were labeled by propidium iodide (PI, 556463, Sigma, USA) [20 (link)]. Briefly, 1 ml microcapsules were placed into a well of 24-well plate containing 300 μl culture medium, 3 μl CMFDA, and 3μl PI and incubated for 10 min. Then the live and dead cells were identified, and photomicrographs were captured under a fluorescence microscope (IX81, OLYMPUS, JAPAN).