Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit 2

For the cell cycle analysis, 48 hrs after transfection, 1 × 10⁶ cells in the log phase of growth were harvested by trypsinization, washed twice with cold PBS, fixed in ice-cold 70% ethanol, and incubated overnight at −20°C. Propidium iodide (PI, 50 μg/ml; Sigma-Aldrich) and RNAse A (0.1 mg/ml, Sigma-Aldrich) were added to the cells and allowed to stain for 30 min. Then, cells were examined with a fluorescence-activated cell sorting (FACS) flow cytometer (BD Bioscience, San Jose, CA, USA). DNA histograms were analysed with CellQuest software (BD Biosciences).
For the cell apoptosis assay, an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit II (BD Biosciences) was used to measure the membrane redistribution of phosphatidylserine. Cells were treated per the manufacturer's instructions. The pre-labelled cells were detected and apoptosis was quantified using a FACSCalibur flow cytometer with Cell-Quest software (BD Biosciences). AnnexinV-FITC and PI double stain was used to evaluate the percentage of apoptotic cells. AnnexinV⁻ and PI⁻ cells were used as controls. AnnexinV⁺ and PI⁻ cells were considered apoptotic, and AnnexinV⁺ and PI⁺ cells were defined as necrotic. The percentage of cells with apoptotic nuclei (% apoptosis) was calculated. Each test was repeated in triplicate.

Twenty-four hours after exposure to each agent, the NP cell–alginate 3D composites were dissolved in 55-mM sodium citrate at 4°C until the beads released the cells, which were collected by centrifugation. The NP cells were washed twice in phosphate-buffered saline, counted (3.6 × 10⁵), and labeled with the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit II (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Both early apoptotic cells (FITC+/PI−) and late apoptotic cells (FITC+/PI +) were monitored with a flow cytometer (FACS Cant; BD biosciences, CA, USA). The present study included both early and late apoptotic cells, and the results of both groups of cells were combined in the analysis [11] (link), [16] (link), [17] (link).

To further characterize cell death after thermal stress, we used the Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit II (BD Biosciences, San Jose, CA, USA). Immediately afterwards, cells were analyzed in a FACS (fluorescence-activated cell sorting) Canto II Flow Cytometer (BD Biosciences) at the FACS core facility of the University Medical Center Göttingen (UMG). For each condition, 10,000 cells were counted. Analysis was done using FlowJo Single Cell Analysis Software v10 (FlowJo LLC, Ashland, OR, USA).