Annexin 5 apc

After treatment, NRK-52E cells were washed with precooled phosphate buffered saline and costained with Annexin V-APC and propidium iodide (MultiSciences Biotech Co., Ltd). Apoptosis was evaluated using a BD FACSCalibur flow cytometer.

The extent of apoptosis was determined by flow cytometry using an Annexin V-APC (Annexin V-Allophycocyanin)/7AAD (7-Aminoactionomycin) assay (Multi sciences biotech, Co., Ltd, Hangzhou, China). According to the manufacturer’s instructions, after co-culture with non-irradiated/irradiated BV2 cells, HT22 cells were washed twice with cold PBS, resuspended, and incubated with Annexin V-APC (dilution 1:40) and 7AAD (dilution 1:40) in the dark for 15 min at room temperature. For each experiment, 1 × 10⁵ cells were analyzed.

Cytarabine (Ara-C) was provided by Target Molecule Corp, China. SC75741 (a NF-κB inhibitor) was offered by Target Molecule Corp, China. Puromycin was purchased from Solarbio (Beiijng, China). RPMI 1640 medium was provided by GE Healthcare Life Sciences HyClone Laboratory (Logan, UT). Annexin V-APC and 7-ADD were purchased from Multi Sciences, China. Matrigel Matrix was purchased from Corning Biocoat, China. Anti-Nrf2 antibody (#ab89443) came from Abcam (Cambridge, UK). The anti-RFC4 antibody was purchased from Gene Tex (#N1C3), USA, whereas the anti-phospho-c-Jun (#2361), anti-phospho-JNK (#4668), anti-c-Jun (#9165), and anti-JNK (#9252) antibodies were provided by Cell Signaling Technology (Danvers, MA, USA). Anti-NF-κB p65 (#AF5006) and anti-phospho-NF-κB p65 (#AF2006) were offered by Affinity Biosciences, USA. Anti-β-Actin antibody was acquired from Solarbio Life Sciences, China. CoraLite594-conjugated Goat Anti-Rabbit lgG (H + L) was obtained from Proteintech, China.

Cells were harvested with trypsin without EDTA, washed three times with phosphate-buffered saline (PBS), and resuspended in annexin binding buffer at a concentration of 1 × 10⁶ per 100 μl. Then, cells were stained with 5 μl annexin V-APC and 10 μl 7-aminoactinomycin D solution (MultiSciences, Hangzhou, China), and incubated at room temperature for 5 min. Finally, cells were separated by flow cytometry (FACSCalibur), and analyzed with Flowjo V10 software.

The trypsinized A549/NCI-H1975 cells were adjusted to 3 × 10⁵ cells/mL with serum-containing DMEM/RPMI-1640 medium. The Annexin V-APC and the 7-AAD Apoptosis Detection Kit (MultiSciences, China) were used in the cell apoptosis assay. The A549 or NCI-H1975 cells were transfected with miR-1976 mimic, miR-1976 inhibitor, Lv-NCAPH lentivirus, or sh-NCAPH lentivirus for 24 h. Then, the cells were treated with serum-free DMEM/RPMI-1640 medium for 24 h to induce apoptosis. Next, the cells were stained with Annexin V-APC and 7-AAD for 30 min in the dark. Cell cycle detection was performed using cell cycle staining buffer (MultiSciences, China). Cell cycle detection was conducted when the treated cells were washed with PBS and then stained with cell cycle staining buffer for 30 min. The samples were finally tested using a flow cytometer(Becton Dickinson), and the apoptosis populations were calculated by FlowJo X software. Similarly, the cell cycle was analyzed using FlowJo X software.

Cells transfected with control or SPATS2L KD lentiviral fluid were collected after transfection 96 h. For apoptosis analysis, cells were stained with PI and Annexin-V-APC for 15 min in a binding buffer (MULTI SCIENCES, China). For the differentiation test, CD11b FITC (BD, USA) and CD14 FITC (BD, USA) were used. Cells were stained with CD11b or CD14 antibody for 30 min in 1xPBS. All the processed cells were analyzed using a NovoCyte D2060R flow cytometer (ACEA, China). GFP-positive cells were sorted by Beckman moflo Astrios EQ.