Muse count viability kit

Human MDA-MB-231 and MCF-7 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA). Leibovitz’s L-15 medium, RPMI-1640 medium, Fetal Bovine Serum (FBS), Penicillin-streptomycin Cocktails and Cystatin C (Catalog number: PHP0044) were obtained from Thermo Scientific (Rockford, IL). SAHA was purchased from Sigma-Aldrich (St. Louis, MO). Muse Cell Cycle kit, Muse Annexin & Dead Cell kit, and Muse Count & Viability kit were from Millipore (Darmstadt, Germany). Human MAPK Antibody Array kit was purchased from R&D Systems (Minneapolis, MN). High Pure RNA Isolation kit and Transcriptor First Strand cDNA Synthesis kit were obtained from Roche Diagnostics GmbH (Mannheim, Germany). Exprofile Human autophagy Gene qPCR Array kit was obtained from Genecopoeia (Rockville, MD). Power SYBR Green PCR Master mix, RIPA Cell Lysis buffer and BCA Protein Assay kit were from Life Technologies (Austin, TX). Polyclonal anti-beclin-1 antibody, polyclonal anti-Atg3 antibody, polyclonal anti-Atg5 antibody, polyclonal anti-Atg7 antibody, polyclonal anti-Atg12 antibody, polyclonal anti-Atg16 antibody, polyclonal anti-Atg4A antibody, polyclonal anti-Atg4B antibody, polyclonal anti-Atg9B antibody, polyclonal anti-LC3II antibody were obtained from Abcam Inc (Cambridge, MA). Protease inhibitor and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Viability studies were performed 24 h after O₃ treatment by measurement of LDH release and cytofluorimetric assay as previous described [16 (link)]. The LDH levels in the supernatant were calculated base on the kit instructions (EuroClone Milan, Italy). All tests were performed in triplicate and assays were repeated five times independently with average results reported.
Cytofluorimetric assay was performed using Muse Count & Viability Kit (Millipore, Corporation, Billerica, MA, USA). Briefly, cells (1x10⁶ to 1x10⁷ cells/ml) were suspended in PBS. Then, 380 μl of Muse Count & Viability working solution was added to the cells, and 20 μl of this cell suspension was incubated for 5 minutes at room temperature in the dark. Cells were analyzed by using a Muse Cell Analyzer.

TMD8 cells were transfected with constructs of WT BTK or BTK^C481S mutants using kit V, Program U-13 on Amaxa Nucleofector, according to the manufacturer's protocols (Amaxa, Cologne, Germany). After transfection, the cells were co-cultured with NKTert cells in a 24-well plate for 24 hrs for recovery. Ibrutinib, cerdulatinib and vehicle (DMSO) were then added into the transfected TMD8 cells and cellular viability was determined with Muse™ Count & Viability kit using Muse Cell Analyzer (Millipore, Hayward, CA, USA). The cell survival was determined by flow cytometry using the Annexin V/7-AAD Apoptosis Detection Kit I on freshly isolated CLL cells following manufacturer's instructions (BD Biosciences).