Mebcyto apoptosis kit

Transfected A375 melanoma cells were plated in six-well plates until 90% confluence and then treated with cytotoxic agents overnight. The cells were cultured with etoposide (from 1 to 100 μM) or gemcitabine (from 10 to 100 μM). Cell death was determined by Annexin V and PI staining in accordance with the manufacturer’s instructions (MEBCYTO^® Apoptosis Kit; Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), and analyzed by flow cytometry.

Murine pancreatic tumor was harvested and chopped into small pieces, and digested in 0.25% Trypsin EDTA (Thermo Fisher Scientific) at 37 °C for 15 min. After neutralizing with fetal bovine serum (FBS), samples were further digested with 2 mg/ml collagenase A (Roche) and DNase I (50 U/ml, Worthington, Columbus, OH) at 37 °C for 20 min. Following multiple washes with PBS supplemented with 2% FBS, cells were filtered through a 40 µm cell strainer. Red blood cells were lysed with ACK Lysing Buffer (Thermo Fisher Scientific) after passing through a strainer. Filtered single cells were incubated with anti-CD11b (Thermo Fisher Scientific), F4/80, Ly6C and Ly6G antibodies (Biolegend, San Diego, CA). LIVE/DEAD Fixable Dead Cell Stain Kit (Thermo Fisher Scientific) was used to exclude dead cells. Cells were analyzed by BD FACSAria II (BD Biosciences, Franklin Lakes, NJ). For cell apoptosis assay, MEBCYTO apoptosis kit (Medical Biological Laboratories, Nagoya, Japan) was used according to manufacturer’s protocol and analyzed using Guava easyCyte plus (Luminex, Austin, TX, USA).

Cell proliferation was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (24 (link)). The MTT absorbance was examined at 570 nm in a SpectraMax Plus 384 Microplate Reader (Molecular Devices, LLC, USA) at 24, 48, 72, 96, and 120 h after treatment. The apoptosis rate of cells was assessed using a MEBCYTO Apoptosis Kit (Medical and Biological Laboratories Co., Ltd., Aichi, Japan) according to manufacturer’s instructions. Briefly, 1 × 10⁵ cells were stained with Annexin V-fluorescein isothiocyanate and propidium iodide (PI) for 25 min after trypsinization and then analyzed with flow cytometry (BD FACSCalibur, Becton Dickinson, San Jose, CA, USA). The apoptosis rate was determined as the ratio of Annexin V-positive apoptotic cells in all counted cells.

Cells were seeded at 4 × 10⁵ cells per well in 6-cm dishes and exposed to control (DMSO), 20 µM RARγi-1 or 50 µM RARγi-2 for 24 h. Cells were harvested and fixed with 70% ice-cold ethanol for 1 h for cell cycle analyses. The cells were then treated with RNase A (100 µg/ml) and stained with propidium iodide (50 µg/ml) (Dojindo Laboratories, Kumamoto, Japan). For apoptosis analyses, after seeding and drug exposure, cells were harvested and stained with propidium iodide and annexin V-FITC using a MEBCYTO® Apoptosis Kit (4700, Medical & Biological Laboratories, Tokyo, Japan) according to the manufacturer’s instructions.
The cell cycle and apoptosis status were analyzed using a FACS Verse (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).