Annexin 5 fitc cell apoptosis assay kit

Butaprost and AH6809 were purchased from Caymen (USA), Recombinant human TGF-β1 was purchased from PeproTech (UK), The CCK-8 kit and Trizol RNA extracting kit were purchased from Invitrogen (USA). Mouse cAMP and PGE2 ELISA kit were purchased from Weston Biology Company (Shanghai, China). The reverse transcription (RT) kit was purchased from Fermentas (USA). The real-time quantitative PCR (RT-qPCR) kit was from Roche (USA). The Annexin V-FITC cell apoptosis assay kit was purchased from Beyotime (Shanghai, China). Nephrin, podocin, and CD2AP primers were purchased from Invitrogen (USA). Rabbit anti-mouse polyclonal antibodies for Nephrin, podocin and caspase3 were purchased from Abcam (UK). Rabbit anti-rat monoclonal antibodies of GAPDH, CD2AP, PI3K-p85 and Akt were purchased from Cell Signaling (USA), as well as phosphorylated PI3K-p85 and Akt monoclonal antibodies. The horseradish peroxidase (HRP) labeled goat anti-mouse and goat anti-rabbit IgG secondary antibody were purchased from SantaCruz (USA). Alexa Fluor 488 labeled goat anti-rabbit IgG (H+L) was purchased from Fcmacs (Nanjing, Jiangsu province, China).

TUNEL (TdT-UTP nick end labeling) analysis was executed by the one step TUNEL kit (#TUNEL-A2, Dingguo, Beijing, China). Concisely, the cells were fixed by 4% paraform, added 0.1% Triton X-100 for 3 min on ice, incubated with TUNEL for 60 min at 37 °C. The apoptotic cells revealed green fluorescence. The cell apoptosis rates were also detected with flow cytometry. Briefly, 1.0 × 10⁵ cultured cells were harvested by trypsinization, washed with phosphate buffer saline (PBS) and trypsinized into single cell suspensions. Then, the cells from each sample were stained with annexin V-FITC and propidium iodide (PI), and processed for Annexin V FITC/PI apoptosis detection (Becton Dickinson, New York, NY, USA) according to the manufacturer’s instructions. annexin V-FITC cell apoptosis assay kit was purchased from Beyotime (#C1063, Shanghai, China). Experiments were carried out in triplicate.

Cell apoptosis was measured using Annexin V-FITC cell apoptosis assay kit (Beyotime, China). Cells were trypsin-treated (BD Biosciences, USA) and washed with cold PBS. Cells were centrifuged and then resuspended in 195 μl of 1× Annexin V loading buffer after the supernatant was removed. Subsequently, cells were incubated with 5 μl Annexin-V/FITC, followed by 10 μl PI for 15 min at room temperature in the dark. The results were analyzed by CellQuest software (BD Biosciences, NJ, USA) of FACS Calibur flow cytometry (Becton Dickinson, CA, USA).

C6 cells were seeded in 6-well plates for 48 h and then treated with different treatments for indicated times. The cell suspension was then prepared and stained with an Annexin V-FITC Cell Apoptosis Assay Kit (Beyotime Biotechnology, Shanghai, China) according to the instruction book. Apoptosis flow cytometry was carried out on a Novocyte Instrument using Novo Express software (ACEA, China), and the stained cells were examined by a Leica Microsystems microscope.

Cell apoptosis was evaluated by flow cytometry using Annexin V-FITC cell apoptosis assay kit (Beyotime, Shanghai, China). HK-2 cells after treatment with 5 μg/ml of LPS for 8 h were collected using trypsin-EDTA (Thermo Fisher Scientific) and brief centrifugation (1000×g for 5 min at room temperature). 100,000 cells resuspended in PBS were then collected by centrifugation and were subject to Annexin V-FITC/PI double staining following manufacturer’s instructions. Cells with Annexin V-FITC⁺ PI⁻ staining were considered apoptotic cells (Schutte et al. 1998 (link)). The apoptotic result is calculated as Q2 + Q3, where Q2 is early cell apoptosis, and Q3 is late apoptosis. Our figures are drawn based on adopting the approach to calculating the percentage of Q2 + Q3.

The following antibodies were used in this study: anti-CNTF Ab (Abcam Cambridge, MA, USA, Cat. # ab46172), anti-BAX Ab (Abcam Cambridge, MA, USA,Cat. # ab32502), anti-BCL2 Ab (Abcam Cambridge, MA, USA, Cat. # ab117115), anti-Ki67 Ab (Abcam, Cambridge, MA, USA, Cat. # 16667), anti-Wee1 Ab (Cell Signaling, Beverly, MA, USA, Cat. # 4938 S), anti-AKT Ab and anti-p(Ser473)-AKT Ab (Cell Signaling, Beverly, MA, USA, Cat. # 2920 S and 4060 P), anti-cleaved Caspase 3 Ab (Cell Signaling, Beverly, MA, USA,Cat. # 9664), anti-CNTFRα Ab (Santa Cruz, USA. Cat. # sc-9993), goat anti-mouse IgG Fluor488 Ab, goat anti-rabbit IgG Fluor 546 Ab (Invitrogen, NY, USA, Cat. # A11017 and A11071), anti-GAPDH Ab and mouse and rabbit secondary Ab (Kangcheng, Shanghai, China. Cat. # KC-5G4, KC-MM-035, KC-RB-035).
Other reagents and kits used were: CCK8 (Dojindo, Japan, Cat. # CK04), Annexin V-FITC cell apoptosis assay kit, Cell cycle assay kit (Beyotime, Shanghai, China, Cat. #C1063and C1052), LY294002 (Cell Signaling, Beverly, MA, USA, Cat. #9901), 5-Aza-2-dC (Sigma Aldrich, Saint Louis, MO, USA, Cat. #A3656), CNTFRα siRNA (Santa Cruz, USA, Cat. #35076), SYBR real time kit (Takara, Dalian, China, Cat. #DDR041A), MSP-PCR kit (Epigentek, Farmingdale, NY, USA, Cat. # P-1016-80).