Apc annexinv dead cell apoptosis kit

Apoptotic cells were detected using an Apoptosis kit (APC-AnnexinV/Dead Cell Apoptosis Kit, (Invitrogen, Life Technologies). The cells were plated in 6-well plates at the density of 0.5 × 10⁶ cells/well and incubated for 24 h. Then, ACF was added to the cells (5 µM)^{20 (link)} and incubated for an additional 24 h. The negative control was prepared in medium with no additives. Briefly the cells were then incubated for 15–20 min in the dark at RT in 100 μL of Annexin-binding buffer 5X (10mMHEPES pH7.4, 140 mM NaCl, 2.5 mM CaCl2), containing 5 μL of Annexin V-APC and 1 μL of Propidium Iodide (PI) solution at 100 μg/mL, and then analyzed by FACS (FACS Calibur, Becton-Dickinson, Franklin Lakes, NJ, USA) within 1 h. Subsequent analyses were performed using FlowJo software (FlowJo LLC, Ashland, OR, USA).

The proportion of HSPCs in G0/G1, G2/M, and S phases was analyzed using EdU incorporation assay over 24 h using Click-it EdU Alexa Fluor 647 Cell Flow Cytometry Assay Kit and Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. DNA content was detected with 7-AAD. To distinguish between cells in G0 and G1 phases, BM cells were stained with Hoechst 33342 at 37 °C. After 45 min, 1 μg/ml PY was added and cells were incubated at 37 °C for 45 min^{57 (link)}. Samples were analyzed by FACSAria flow cytometry. Apoptotic cells were analyzed using the APC-Annexin V/Dead Cell Apoptosis Kit (Invitrogen) according to the manufacturer’s protocol. Briefly, BM cells were treated with reagents provided in the kit and analyzed by FACS Aria flow cytometry and quantified using the FlowJo software.

At 24, 48, 72, or 96 hrs after treatment with ribavirin (50 μM), cells were collected, washed in PBS and prepared for flow cytometry. For cell death, we used reagents from the APC-AnnexinV/Dead Cell Apoptosis Kit (Invitrogen). For cell cycle analysis, cells were labeled with FITC-anti-Ki67 antibody (Abcam, Cambridge, MA, USA) and run on a flow cytometer (FACS Calibur, Becton-Dickinson, Franklin Lakes, NJ, USA). Subsequent analyses were performed using FlowJo software (FlowJo LLC, Ashland, OR, USA).