Aposcreen annexin 5 apoptosis kit fitc

Thymocytes, BaF/3, or L1.2 cells (5 × 10⁵) were incubated (37°C, 5% CO₂) with 10 µg/ml control LVP or FasL-LVP (at different time points, 37°C). Cells were stained with AV-FITC and with PI following the manufacturer protocols (Aposcreen Annexin V apoptosis kit-FITC; Southern Biotech, AL, USA). All samples were analyzed in a Gallios cytometer. The number of early apoptotic (AV+/PI−) and necrotic/late apoptotic (AV+/PI+) cells was expressed as a percentage of total cells.

To study cell viability, hCPC were detached with trypsin–EDTA 48 h post-transfection, labeled with DAPI (1/1000; Sigma-Aldrich) and quantified by flow cytometry on a FACS Canto 3L flow cytometer (BD Biosciences, San Jose, CA). For proliferation assays, 5-ethynyl-2’-deoxyuridine (EdU; 10 μM) was added to hCPC cultures 12 h prior to analysis. Proliferating cells were detected with the Click-iT Flow-Cytometry Kit (Thermo Fisher Scientific). For apoptosis analysis, cells were exposed to H₂O₂ (500 μM, during 5 h), then collected (including detached cells) and labeled at 4ºC for 15 min with AnnexinV-FITC (diluted 1:10) in the binding buffer provided by the manufacturer (ApoScreen® Annexin V Apoptosis Kit-FITC; Southern Biotech, Birmingham, AL). Labeled cells were washed with PBS/0.01% BSA and resuspended in 390 μL of binding buffer. Propidium iodide (50 μg/ml, Beckman Couler, Nyon, Switzerland) was added (1:40 dilution) for dual-staining and cells were analyzed by flow cytometry. DAPI and AnexinV/PI positive-cells were quantified on a FACS Canto 3L flow cytometer (BD Biosciences). When indicated, necrosis of hCPC was induced by a short heat treatment (10 min, 60ºC) of attached monolayers.

To measure DNA content, cells were fixed using 70% ethanol and stained with propidium iodide (PI). Utilizing BD accuri C6 Plus (BD Biosciences, San Jose, CA, USA), the sub-G1 population, representing apoptotic cells, was evaluated. For another apoptosis analysis, ApoScreen Annexin V Apoptosis Kit-FITC (Southern Biotech, Birmingham, AL, USA) was used. Cells were stained with both Annexin V-FITC and PI and then, analyzed using a flow cytometer. Total apoptotic cells were determined as the sum of Annexin V⁺/PI^- (early apoptotic) cells and Annexin V⁺/PI⁺ (late apoptotic) cells.