Annexin 5 dead cell kit

After MRI scan on day four after radiation therapy, all mice were euthanized by cervical dislocation and tumors collected for ex vivo analysis. Tumors were harvested and stored in MACS tissue storage solution until dissociated with Tumor Dissociation kit (Miltenyi Biotec, Germany) using a gentleMACS™ Octo Dissociator (Miltenyi Biotec, Germany). Dissociation was performed following the manufacturer’s standard protocol. Cells were washed and diluted into single cell suspensions before assay detection, and red blood cells lysed using VersaLyse™ Lysing Solution, according to manufacturer’s protocol (Beckman Coulter, Brea, Californien, USA). Detection of apoptotic cells, apoptotic stage, and dead cells in tumor tissue was obtained using MUSE® Cell Analyzer and accompanying Annexin V & Dead Cell Kit (Merck Millipore, Darmstadt, Germany) [23 ].
Results of the cellular counts are given in percentages of the gated cells and in number of total cells counted in the gated area. Gates were set in a default setting on a test sample of tissue, and kept fixed for all samples in data set. Results are presented as percentage of all apoptotic cells, including both cells gated as early apoptotic and late apoptotic.

The cell cycle and apoptosis was determined using the Muse™ Cell Analyzer and Annexin V & Dead Cell Kit (EMD Millipore) according to the manufacturer’s instructions. For cell cycle assay, PC12 cells were plated at a density of 1 × 10⁶ cells/mL in 24 well plate and were treated with phlorotannins for 1 h before stimulation with Aβ_25-35 for 24 h. Then, cells were harvested by trypsinization, washed with cold PBS, and centrifuged at 300× g for 5 min at RT. The cell pellets were fixed with 70% ethanol (v/v) for 3 h at −20 °C. The supernatant was discarded and cell pellets (5 × 10⁵) were re-suspended in Muse™ Cell Cycle reagents and incubated for 30 min at RT in the dark. After incubation, the results were examined by the Muse™ Cell Analyzer (Millipore, Billerica, MA, USA).
To determine cellular apoptosis, the cell suspension were treated with Annexin V/dead reagent and incubated in the dark for 20 min at RT. Then stained samples were analyzed with Muse™ Cell Analyzer. The flow cytometry data was obtained from 5000 events (gated cells) per sample. The percentages of cells shown in the figures were calculated from the mean fluorescence intensity in each of the four quadrants. In addition, the coefficient of variation from the mean fluorescence was less than 10%.

Apoptosis was assessed using Annexin V Dead Cell Kit (Merck KGaA, Darmstadt, Germany). After 24 h incubation with test compounds, cells at a density of 3 × 10⁵/mL were trypsinized by means of 0.25% trypsin-EDTA solution (Corning, Manassas, VA, USA) and centrifuged. Cells were then stained for 20 min in the dark, at room temperature, by adding 100 µL of annexin V-PE/7-AAD. The fluorescence intensity was analyzed on a Muse analyzer (Merck-Millipore, Burlington, MA, USA). The experiment was repeated 3 times.

Cell viability and Apoptosis initiation was determined using the MUSE™ Annexin V & Dead Cell Kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. Briefly, after treatment with MSI-1436 and exposure to tunicamycin, all treated and untreated Hep-G2 cells were collected by trypsinization and suspended in HBSS containing 1% FBS. Then, cells were stained with the Annexin V & Dead Cell Kit for 20 minutes at room temperature and analyzed using the Muse cell analyzer (Merck Millipore, Darmstadt, Germany). The apoptotic ratio was calculated by the identification of four populations: (i) non-apoptotic cells, not undergoing detectable apoptosis: Annexin V (-) and 7-AAD (-); (ii) early apoptotic cells, Annexin V (+) and 7-AAD (-); (iii) late apoptotic cells, Annexin V (+) and 7-AAD (+); (iv) cells that died through non-apoptotic pathway: Annexin V (-) and 7-AAD (+).