Annexin 5 binding buffer

Leukocytes were added to a 5mL round bottom tube (BD Falcon) at a density of 5x10⁵ cells and centrifuged at 350 x g for 8 min at 4°C. Cells were washed twice with 1x Annexin V Binding Buffer (BD Biosciences, Cat# 556454), resuspended in 200 μL 1x Annexin V Binding Buffer, and incubated for 30 min in the dark with 5 μL FITC Annexin V (BD Biosciences, Cat# 560931) and 4 μL propidium iodide (Sigma, Cat# P4864) diluted 1:10 in 1x Annexin V Binding Buffer. Finally, leukocytes were washed with 500 μL of 1x Annexin V Binding Buffer and fixed with 1% formaldehyde. Prior to analysis, leukocytes were washed twice with 1 x PBS^-/^-, centrifuged at 350 x g for 5 min at 4°C, and the supernatant was decanted. Data was acquired using ImageStream Mk II Imaging Flow Cytometer (Amnis) and analyzed using IDEAS Image Data Exploration and Analysis Software (Amnis). A minimum of 1x10⁴ events was acquired. Leukocytes were gated based on the normalized frequency of a fluorescent minus one sample. The reported protocol isolates skin leukocytes with ~ 90% viability (Figure 1).

For the FCM analysis of apoptosis, 1.0–5.0 × 10⁶ cultured cells were collected by trypsin digestion without EDTA and washed twice by cold PBS. After centrifuged at 800 rpm for 5 min, cells were resuspended in 200 μL 1xAnnexin V Binding Buffer (BD Pharmingen) and stained under dark with 2 μl APC -conjugated Annexin V antibody (BD Pharmingen) for 30 min. After being washed twice, cells were resuspended in 200 μL of 1xAnnexin V Binding Buffer again and added with 5 μL 7-AAD to incubate for 15–20 min, which were finally examined by BD LSRFortessa X-20 flow cytometer (San Diego,CA).
For the FCM analysis of the cell cycle, trypsinized cells were fixed overnight in 70% ethanol at -20 °C, washed with staining buffer, and stained with an anti-phospho-histone H3 (PHH3) antibody (Alexa Fluor® 488 Conjugate; Cell Signaling Technology, 1:100) at 4 °C for 30 min. DNA was subsequently stained with 40 μg/ml propidium iodide (PI) solution for 15–20 min. The cells were finally examined by flow cytometry. All of the acquired flow cytometry data were analysed with FlowJo 10.0 or ModFit LT5.0 software. To dynamically study the process of tumour cell cycle progression, we blocked A549, NCI-H1299 and HeLa cells at the G1/S or G2/M phase by double thymidine or RO3306 blockade as previously mentioned, followed by release into fresh medium for various time periods and subsequent cell cycle analysis.

CHL-1 or 624-mel cells were seeded in cRPMI at a density of 70,000 cells per well in a 12-well plate. After overnight incubation, cells were treated with different cell death inducers at indicated concentrations. The percentage of FBS in the culture medium for this assay was lowered to 2.5%. After 24 h, 48 h or 72 h viable, dying and dead cells were harvested. After washing the cells with PBS, single cells were resuspended in 100 µL composed of 2 µL AnnexinV-APC (1/50; BioLegend, Amsterdam, Netherlands; 640920), 3.3 µL DAPI solution (0.03 µg/mL) and 94.7 µL 1X Annexin V Binding Buffer (BD Biosciences, 55645). Cells were incubated for 20 min at 4 °C and in the dark. After incubation, 300 µL of 1X Annexin V Binding Buffer was added and cells were immediately analyzed using flow cytometry (BD LRS Fortessa). Flow cytometric data were analyzed with FlowLogic software (Miltenyi Biotec, Version 7.3).