Muse annexin 5 dead cell assay

MTS assay CellTiter 96 AQueous One Solution Proliferation Assay, Dead End Fluorometric TUNEL system and Caspase-Glo 3/7 assay purchased from Promega, (Madison, WI, USA). Click-it TUNEL Alexa Fluor 594 from Life Technologies. Muse Annexin V & Dead Cell Assay (Merck, Millipore, USA). LDH-Cytotoxicity Assay was purchased from Thermo Scientific (Thermo, Waltham, MA, USA). Staurosporine (STS), propidium iodide (PI) and Cisplatin were obtained from Sigma-Aldrich (St. Louis, MO, USA). The cell culture media (RPMI1640, DIMEN) and heat inactivated fetal bovine serum (FBS) as well as the antibiotics (penicillin and streptomycin) were purchased from Corning CellGro (New York, NY, USA).

The number of viable cells, type of cell, and cell death, and stage of apoptosis of mHCN2-expressing hMSCs after selection with geneticin were analyzed using the Muse® Annexin V & dead cell assay (Merck Millipore) following the manufacturer’s instructions. This assay allows quantitative identification of live, early and late apoptotic, and dead cells by measuring the intensity of cell fluorescence. Briefly, cells transfected with the mHCN2 gene, only, with the pIRES vector, and non-transfected hMSCs were harvested with trypsin-EDTA and suspended in DMEM containing 10 % fetal calf serum. Cell suspension (100 μl) and Muse™ annexin V & dead cell reagent (100 μl; Annexin V and 7-AAD) were thoroughly mixed. Samples were incubated for 20 min in the dark. The Muse™ Cell Analyzer (Merc Millipore) was used to measure cell fluorescence. Cells were separated into groups according to the intensity of green (Annexin; early and late apoptotic cells) and red (7-AA; dead cells) fluorescence. Cell viability was tested 5 days after the transfected cell growth with 50 μM geneticin. Non-transfected cells were grown in parallel for the same time period.

Cells (2 × 10⁵) were seeded in 100‐mm culture plates and, after 24 h, were treated with 5 μM olaparib or 5 mM metformin for 2 days. Afterward, cells were detached, centrifuged at 300 g for 5 min, and resuspended at a density of 5 × 10⁵ cells/ml in 1× PBS‐1% FBS. Then, 100 μl of the cell suspension was mixed with Annexin‐V kit buffer (1:1; Muse™ Annexin‐V & Dead Cell Assay, Merck Millipore, Merck KGaA, Darmstadt, Germany). After 20 min of incubation at room temperature, cells were examined in a Muse™ Cell Analyzer (Merck Millipore). All conditions were assessed in two independent experiments.

Live, dead, early-, or late-apoptotic Caco-2 cells were quantified using the Muse Annexin V & Dead Cell Assay (Merck) as specified. Briefly, cells (2 × 10⁴ cells/well) in 24-multiwell plates were incubated for 24 h with Alexa-Fluor 555 labeled PT-gliadin (1 µg/µL). Then, cells were washed three times with PBS, trypsinized, resuspended in 1% BSA (v/v), and 1 volume of Annexin V reagent was added and finally incubated at room temperature for 20 min and next analysed in triplicates using the Muse Cell Analyzer (Merck); for autophagy detection, the Muse Autophagy LC3-antibody based kit (Merck) was adopted: in this case, after fluorescent PT-gliadin incubation, cells were permeabilized and incubated on ice for 30 min with the anti-LC3 mouse monoclonal Alexa Fluor 555 conjugated antibody, according to the manufacturer’s protocol. Then, the intracellular LC3 fluorescence was assayed in triplicates using the Muse Cell Analyzer (Merck).