Annexin 5 fitc apoptosis kit

Percentage of apoptotic cells was examined immediately after culture in fresh cells using Annexin V-FITC Apoptosis Kit (BioVision, Inc., Milpitas, CA, USA) according to manufacturer instructions on a Nikon Eclipse Ti (Nikon, Tokyo, Japan) fluorescent microscope equipped with standard filters. Then, 500,000 cells were resuspended in binding buffer and 5 μL of Annexin V-FITC and propidium iodide were added. Cells were incubated for 5 min in the dark (room temperature), and then were transferred to a glass slide and covered with a glass coverslip. Cells were observed under 40× magnification using corresponding filters. Annexin V binds to phosphatidylserine, which undergoes externalization during apoptosis; cells stained with annexin were recognized as an apoptotic, otherwise as not. The total amount of cells counted was 200, and the percentage of apoptotic cells was then calculated [28 (link)].

To detect cell cycle, cells were stimulated with anti-CD3 antibody (5 μg ml⁻¹) and anti-CD28 antibody (2 μg ml⁻¹) for zero, one, two and three days. Collected cells were suspended in ice-cold 90% methanol and incubated at −30 °C overnight for fixation and permeabilization. Fixed and permeabilized cells were re-suspended in propidium iodide (PI)-containing buffer and DNA content at indicated time points was analysed by flow cytometry. Cells in the G2/M phase, S phase and G0/G1 phase were detected as PI^high, PI^int and PI^low-stained cells, respectively.
To detect apoptosis, cells were stimulated with anti-CD3 antibody (5 μg ml⁻¹) and anti-CD28 antibody (2 μg ml⁻¹) for 60 h. Apoptotic cells were stained by AnnexinV-FITC Apoptosis kit (BioVision) and analysed by flow cytometry.