Annexin 5 apc pi kit

To assess the cell cycle, P6 MSCs treated with and without A83-01 were trypsinised, pelleted and fixed/permeabilised in ice-cold 70% ethanol for 30 min at 4 °C. The cells were centrifuged at 2000 rpm for 5 min at 4 °C and washed with PBS. The pelleted cells were incubated with 50-µL RNase (100 µg/mL, # R4875, Sigma) at room temperature (RT) for 15 min to remove any RNA contamination. Propidium iodide (PI) (200 µL, 50 µg/mL, Sigma) was added and samples analysed immediately by flow cytometry using BD FACS Canto^TM II using the linear mode for PI. The data were analysed using the Dean-Jett-Fox model in FlowJo 7.6.3 [46 (link),53 (link)].
To assess apoptosis following treatment, P6 MSCs were trypsinised and stained with an Annexin V-APC/PI kit following the manufacturer’s protocols (eBioscience, Waltham, MA, USA). The trypsinised cells were pelleted and washed in 2% foetal bovine serum (FBS)/PBS, followed by washing with binding buffer and centrifuging at 1100 rpm for 5 min at 4 °C. The pellet was incubated with 5-µL Annexin-V-APC in 100-µL binding buffer for 10 min at RT protected from light, followed by washing with binding buffer and resuspending with 5 µL of PI in 200-µL binding buffer. Events were acquired immediately by flow cytometry using BD FACS Canto^TM II and analysed with FlowJo 7.6.3 [46 (link)].

To assess the cell cycle status, P6 A83-01 treated and untreated cells were detached, pelleted and fixed in ice-cold 70% ethanol at 4 °C overnight. They were washed with 2%FBS/PBS and incubated with 50 μl RNAse (100 μg/ml, Sigma) at room temperature for 15 minutes. 200 μl of propidium iodide (PI) (50 μg/ml, Sigma P4170) was added and the cells were analysed immediately by flow cytometry using BD FACS Canto^TM II on PI-linear scales. The data were analysed using Flow Jo 7.6.3.
To assess apoptosis, P6 A83-01 treated and untreated cell-pellets were stained with Annexin V-APC/PI kit following the manufacturer’s protocol (#88–8007, eBioscience). Briefly, cells were trypsinised and resuspended in 100 μl binding buffer, 5 μl of Annexin V-APC solution was added to the cell suspension and incubated for 15 minutes at room temperature protected from light. Following washing with the binding buffer, 5 μl of PI was added to the cells suspended in 200 μl binding buffer and events immediately acquired by flow cytometry using BD FACS Canto^TM II and analysed with Flow Jo 7.6.3.