Annexin 5 pi staining

Annexin V-PI staining was used to evaluate A549 cells apoptosis according to the manufacturer’s instructions (Roche, Germany). Briefly, A549 cells were plated in a 12-well cell culture plates at 2×10⁵ cells per well and pre-cultured in 2.5% FCS-RPMI 1640 with or without 1 µg/ml anti-FST monoclonal antibody for 2 h, and then were incubated in the presence of 10 ng/ml activin A for 24 h. A549 cells were stained by Annexin V-Alexa Fluor-488/PI and the stained cells were analyzed by flow cytometry to determine the percentages of AnnexinV+/PI− (early apoptosis) and AnnexinV+/PI+ (late apoptosis) cells. The experiment was repeated 3 times.

For apoptosis evaluation cells were seeded into six-well plates at a density of 2 × 10⁵ cells/well. Apoptosis detection was performed through the analysis of Annexin V/PI staining (Roche, 11 988 549 001). Flow cytometric analyses were performed on an FC500 flow cytometer (Beckman Coulter).

Cell proliferation was detected by MTT assay (Sigma), cell apoptosis was detected by flowcytometry using Annexin V/ PI staining(Roche company). Cell cycle assay was performed by flowcytometry using PI staining as previously described [13 (link)].