Annexin 5 fluorescein isothiocyanate fitc propidium iodide apoptosis kit

DMEM was provided by HyClone Company (Thermo Fisher Scientific, Waltham, MA, USA). FBS was purchased from Thermo Fisher Scientific. Penicillin-streptomycin, 0.25% trypsin, and cell counting kit-8 (CCK-8) agent were purchased from Beijing Solarbio Science & Technology Company (Beijing, China). siRNA for NKAP was purchased from Gene (Shanghai, China). Lipofectamine 2000 was obtained from Thermo Fisher Scientific. Trizol reagent was provided by Thermo Fisher Scientific. RevertAid First Strand cDNA Synthesis Kit and SYBR Premix Ex Taq II were purchased from Takara (Japan). Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit was ordered from Thermo Fisher Scientific. CoCl₂ was ordered from Sigma-Aldrich Co. (St Louis, MO, USA). RIPA Lysis Buffer, BCA Protein Assay Kit, and Protease Inhibitor Cocktail were all purchased from CwBio (Beijing, China). Primers were synthesized by Genewiz Company (Beijing, China). Transwell cell culture plates were ordered from EMD Millipore (Billerica, MA, USA). Matrigel Matrix was provided by BD Biosciences (San Jose, CA, USA). Protein Marker was obtained from Thermo Fisher Scientific. Enhanced chemiluminescent (ECL) developer was obtained from PTG (Chicago, IL, USA). This article does not contain any studies with human participants or animals performed by any of the authors; hence ethical approval is not required.

Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from HyClone (Thermo Fisher Scientific, Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific. Penicillin–streptomycin, 0.25% trypsin and cell counting kit-8 (CCK8) agent were purchased from Beijing Solarbio Science & Technology Company (Beijing, China). Lipofectamine 2000 was purchased from Thermo Fisher Scientific. Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Kit was purchased from Thermo Fisher Scientific. Radioimmuno-precipitation assay (RIPA) lysis buffer, bicinchoninic acid (BCA) protein assay kit, protease inhibitor cocktail, and all antibodies were purchased from CWBIO (Beijing, China). Primers were synthesized by Genewiz Company (Beijing, China). Transwell cell culture plates were purchased from EMD Millipore (Billerica, MA, USA). Protein marker was purchased from Thermo Fisher Scientific. Electrochemi luminescence (ECL) developer was purchased from Proteintech Group (Chicago, IL, USA).

An Annexin V fluorescein isothiocyanate (FITC)-propidium iodide (PI) Apoptosis kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used for apoptosis analysis. The treated and control cells were collected, and washed twice with PBS at 4°C. Then, the cells were re-suspended in 490 μl binding buffer containing 5 μl Annexin V (Bio-Science, Co. Ltd.) and 5 μl PI. After incubation at 4°C in the dark for 30 min, the cells were sorted using a FACS Aria III (BD Biosciences) and analyzed with BD FACSDiva (version 6.2; BD Biosciences) software. Cells were stained simultaneously with Annexin V-FITC (green fluorescence) and the non-vital dye PI (red fluorescence) allowed the determination of viable cells (FITC^-PI^-), and early apoptotic (FITC⁺PI^-) and late apoptotic (FITC⁺PI⁺), or necrotic cells (FITC^-PI⁺). The rate of apoptotic cells (FITC⁺PI^- and FITC⁺PI⁺) was then calculated according to a previous report (14 (link)).