Pe annexin 5 apoptosis detection kit with 7 aad

Self-renewing macrophages transfected with siRNA were detached from dishes using the enzyme-free cell dissociation buffer and fixed with 70% ethanol. The nuclei were stained with propidium iodide in RNase-containing buffer and analyzed on FACSVerse. Cell cycle (G₀G₁, G₂M, and S) was analyzed using FlowJo software. Cells were also analyzed using the PE Annexin V apoptosis detection kit with 7-AAD (BioLegend) according to the manufacturer’s instructions.

2 × 10⁵ EGI-1 cells/well and 1.5 × 10⁵ HuCCT1 cells/well were plated in 6-well plates. 24 h later, the medium was replaced by fresh culture medium or PAM. 48 h or 72 h later both cells from the supernatant and the plates were collected and stained while using the PE Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, London, UK), according to the manufacturer’s instructions. Flow-cytometric analysis was performed using a Gallios flow cytometer (Beckman-Coulter, Villepinte, France) to calculate the apoptosis rate. The results were analyzed using Kaluza analysis software (Beckman-Coulter).

The four stable cell lines silencing or overexpressing FASN were suspended in Costar® ultra-low attachment 6-well plates (Corning) for 2 days. The PE Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend) and FITC Annexin V Apoptosis Detection Kit with PI were used.

Cells were seeded in 6-well cell culture plates (1.5 × 10⁵ cells/well). The next day, cells were treated with a CDK inhibitor (NVP2 and SY0351 10 nM, YKL-5-124 and THZ531 100 nM) or DMSO (0.0002%). For cell cycle analysis, cells were harvested after 20 h of treatment and resuspended in PBS. Ice-cold methanol was added dropwise while shaking until 70% methanol v/v was reached. Cells were incubated for 2 h at 4 °C and subsequently washed twice with cold PBS. Staining (2 µg/mL Hoechst-33342, 50 µg/mL RNaseA in PBS) was performed for 30 min at 37 °C. The cell cycle distribution was measured in an FACS Canto (BD BioSciences, Heidelberg, Germany) and analyzed using FlowJo™ v10.8 Software (BD BioSciences, Heidelberg, Germany). For apoptosis analysis, PE AnnexinV Apoptosis Detection Kit with 7-AAD (BioLegend, San Diego, CA USA) was used according to the manufacturer’s manual. Measurement was performed in an FACS Canto while analysis was performed in the corresponding BD FACSDiva software^TM (BD BioSciences, Heidelberg, Germany). Two-tailed Student’s t-test was applied for calculation of significance.

For in vitro experiments, young mouse embryonic fibroblasts (MEF) were cultured in 3% O₂. Senescent MEFs were prepared by treatment with 15 ng/ml doxorubicin hydrochloride (Sigma-Aldrich) in 20% O₂ for 7 days. Cells were treated with ABT-263 (LKT Laboratories, Inc.) for 72 h. GM-CSF, IL-6 and IFN-γ (Peprotech) were added to the culture medium during treatment of BM-Mo-MDSCs with ABT-263. Apoptotic cell staining was performed with the PE Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend), according to the manufacturer’s instructions. Apoptotic cells were detected by FACSCalibur (BD Biosciences). For in vivo experiments, wild-type mice were treated with control vehicle (10% ethanol, 30% polyethylene glycol 400 and 60% Phosal 50 PG; Phospholipid Gmbh), or 50 mg/kg/d ABT-263 from day 21 to day 27 after SCT inoculation. At day 28, peripheral blood was collected and stained for Mo-MDSCs (CD11b⁺Ly6C^highLy6G⁻) and PMN-MDSCs (CD11b⁺Ly6C^intLy6G⁺) among CD45⁺ cells.

Apoptosis was determined using the PE Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend) and detected by flow cytometry (BD C6; BD Biosciences, Franklin Lakes, NJ). Briefly, the cells were collected by trypsinization and then washed with PBS three times to remove the debris and complete medium. Subsequently, cells were stained with 5 μl of Annexin V-PE and 5 μl of 7-AAD at dark for 15 min at 23–25°C. For the analysis of the apoptotic cells at different stages as well as the non-apoptotic cells, the early apoptotic cells were defined as positive for PE-Annexin-V and negative for 7-AAD, whereas the late-stage apoptotic cells were defined as positive for PE-Annexin V as well as 7-AAD.

Single-cell suspensions were prepared, counted, and analyzed using specific antibodies (S2 Table) by an LSRII flow cytometer (BD Biosciences), as previously described [91 (link)]. Data were analyzed using the FlowJo software (Tree Star). Apoptotic cells were detected using the PE Annexin V Apoptosis Detection Kit with 7-AAD according to the manufacturer’s protocol (BioLegend) and analyzed using an LSRII flow cytometer.