Cell counting kit-8 (CCK-8) and Annexin V-FITC/PI apoptosis detection kits were purchased from Vazyme Biotechnology Company (Nanjing, China). T-BHP, dimethyl sulfoxide (DMSO), and 2′,7′-dichlorodihydrofluorescein diacetate (DCFHDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cisplatin and N-acetylcysteine (NAC) were purchased from Macklin Biochemical Corporation (Shanghai, China). CpG ODN1826 was purchased from InvivoGen (San Diego, CA, USA). The JC-1 kit was purchased from Beyotime (Shanghai, China). RAW264.7 and AML12 (alpha mouse liver 12) cells were received from American Type Culture Collection (Manassas, VA, USA). Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and Opti-MEM were purchased from Gibco (Waltham, MA, USA). Antibodies for cleaved-caspase 3, caspase 3, caspase 8, cleaved-caspase 8, caspase 9, cleaved-caspase 9, PARP, cleaved PARP, phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, phosphorylated Akt (p-Akt), phosphorylated p38 MAPK, p38 MAPK, phosphorylated JNK, JNK, anti-rabbit IgG, anti-mouse IgG, and GAPDH were all purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-NOX2 antibody (ab80508) was purchased from Abcam (Cambridge, MA, USA). AST and ALT detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
Annexin 5 fitc pi apoptosis detection kit
Manufactured by Vazyme
Sourced in China, United States
The Annexin V-FITC/PI Apoptosis Detection Kit is a laboratory tool used to detect and quantify apoptosis, a type of programmed cell death. It relies on the binding of Annexin V, a protein that has a high affinity for phosphatidylserine, and propidium iodide (PI), a DNA-binding dye. The kit allows for the identification of cells undergoing early and late stages of apoptosis through flow cytometry analysis.
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Lab products found in correlation
Flow cytometry, by BD (8 mentions)
Facscalibur, by BD (7 mentions)
Flow cytometry, by Beckman Coulter (6 mentions)
Cytoflex, by Beckman Coulter (6 mentions)
Facscanto 2, by BD (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Cytoflex s, by Beckman Coulter (4 mentions)
Cell cycle assay kit, by Vazyme (4 mentions)
Tunel brightgreen apoptosis detection kit, by Vazyme (4 mentions)
Cytoflex flow cytometer, by Beckman Coulter (4 mentions)
Cytexpert software, by Beckman Coulter (3 mentions)
Cck 8 kit, by Dojindo Laboratories (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Cleaved caspase 8, by Cell Signaling Technology (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Ldh cytotoxicity assay kit, by Beyotime (2 mentions)
Accuri c6 plus, by BD (2 mentions)
210 protocols using annexin 5 fitc pi apoptosis detection kit
1
Evaluating Oxidative Stress and Apoptosis
2
Quantification of Cellular Apoptosis
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For the assay of apoptosis, the Annexin V-FITC/PI Apoptosis Detection Kits (Vazyme, Nanjing, China) was used. We gathered BEAS-2B cells with trypsin and then washed the cells twice with ice-cold phosphate saline buffer (PBS), and centrifuged the cells at 1,000 g for 5 min. Then, we resuspended the cells in 100 μL binding buffer and incubated with 5 μL of propidium iodide and 5 μL of Annexin V-FITC in dark conditions for 10 min. Afterward, we analyzed the cells using a flow cytometer (Becton Dickinson and Co, Franklin Lakes, NJ, USA). Proportions (%) of dead cells or those apoptosis cells were adopted as rates of apoptosis.
3
Apoptosis detection in mGCs
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The apoptosis of mGCs was determined using Annexin V-FITC/PI apoptosis detection kits (A211, Vazyme, Nanjing, China) under fluorescence-activated cell sorting (FACS) following the manufacturer's protocols. The apoptotic rate of mGCs was calculated and analyzed using FlowJo software (v3.2, Stanford University, Stanford, CA, USA).
4
Measuring BEAS-2B Cell Apoptosis
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Apoptosis of BEAS-2B cells were measured with Annexin V-FITC/PI Apoptosis Detection Kits (Vazyme, Nanjing, China) following manufacturer’s instructions. We collected the treated BEAS-2B from 6-well plates with trypsin, washed twice with pre-cooled phosphate saline buffer (PBS), subsequent to that, centrifugation was carried out at 1800 rpm for a duration of 5 min. Then we re-suspended them with 100 mL binding buffer, and added 5 μl PI staining solution and 5 μl Annexin V-FITC. Cells were kept in the dark for a 10 min incubation period. Finally, we added a 400 mL binding buffer prior to the analysis. Subsequently, the cells were analyzed utilizing flow cytometer (Becton Dickinson and Co, Franklin Lakes, NJ, USA). The apoptosis rates were determined by analyzing the percentage of cells that had either died or were undergoing apoptosis.
5
Evaluating Granulosa Cell Apoptosis
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GC apoptosis was assessed using Annexin V-FITC/PI apoptosis detection kits (A211, vazyme, Nanjing, China) according to the manufacturer’s protocols. Fluorescence activated cell sorting (FACS) was used to detect GC apoptosis. Early apoptotic cells were stained with Annexin V-FITC and late apoptotic cells were stained with propidium iodide (PI). The apoptotic rates of GCs were calculated and analyzed using Flowjo software (v7.6, Stanford University, Stanford, CA, USA).
6
Annexin V-FITC/PI Apoptosis Assay
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LSCC cell apoptosis was examined using Annexin V-FITC/PI Apoptosis Detection Kits (Vazyme, Nanjing, China). LSCC cells were collected and fixed with 4% paraformaldehyde. Cells were further exposed to Annexin V-FITC (5 μl) and PI (5 μl) for 10 min. Apoptotic cells were determined using flow cytometry (ThermoFisher Scientific).
7
Quantifying Cell Apoptosis by Annexin V-FITC/PI
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Cell apoptosis was detected using Annexin V‐FITC/PI apoptosis detection kit (Vazyme), and fluorescence‐activated cell sorter (FACS) was used for quantification. Briefly, 2 × 105 cells were seeded in 6‐well plates and treated with 2 µm TM or DMSO for 48 h. Then, cells were collected and resuspended in 100 μL 1× binding buffer, followed by staining with 5 μL Annexin V‐FITC and 5 μL propidium idodide (PI) for 10 min at room temperature in the dark. The stained samples were analyzed by CytoFLex S (Beckman Coulter, Brea, CA, USA) using cytexpert Software (Beckman Coulter) within an hour. Theoretically, the cells could be divided into the following four groups according to the fluorescence staining through flow cytometry: nonapoptotic cells (Annexin V‐FITC‐negative/PI‐negative), early apoptotic cells (Annexin V‐FITC‐positive/PI‐negative), late apoptotic/necrotic cells (Annexin V‐FITC‐positive/PI‐positive), and dead cells (Annexin V‐FITC‐negative/PI‐positive).
8
Apoptosis Detection in TE-1 and KYSE150 Cells
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The TE-1 and KYSE150 cells were seeded in 24-well plates for 24 h, and the Annexin V-FITC/PI Apoptosis Detection Kit (#A211‑02, Vazyme) was utilized to detect the percentage of cell death according to the manufacturers’ instructions.
9
Salmonella-Induced Macrophage Cytotoxicity and Apoptosis
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Murine RAW264.7 macrophages were seeded in 24-well plates at a density of 2 × 105 cells per well and infected with Salmonella WT and mutants as described above. Cells cultured in DMEM were set as the negative control group, which was not infected with bacteria. After Salmonella infection for 2 h, culture supernatants were harvested for the quantification of lactate dehydrogenase (LDH) with an LDH Cytotoxicity Assay Kit (Beyotime, Shanghai, China). The absorbance value of the DMEM control group was subtracted from all treatment groups according to the manufacturer’s instructions. In addition, cells were collected and centrifuged at 1,000 × g for 10 min and resuspended in 400 μL of binding buffer. Early and late apoptosis among different groups were conducted using an Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, Nanjing, China). Percentages of apoptotic cells were analyzed using a FACS Aria flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with FlowJo_v10 software (FowJo, Ashland, OR, USA).
10
Annexin V-FITC/PI Apoptosis Assay
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Apoptosis was measured using a flow cytometer (Beckman Coulter, FL, USA) and Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, Nanjing, China). Briefly, the cells were placed into 6-well plates, digested with trypsin, and then washed twice with phosphate-buffered saline (PBS). Subsequently, the cells were collected and resuspended in 500 μL of binding buffer, followed by incubation in 5 μL of Annexin V-FITC and 5 μL of PI for 15 min under a light. Finally, the cells were observed and detected by flow cytometry (Beckman Coulter, FL, USA).
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