Cell apoptosis was assessed using annexin-V-FITC and PI staining kit (BD Biosciences). In total, 5×105 cells were collected by trypsinization at 37°C for 5 min, centrifuged under 200 × g at 4°C for 5 min and washed twice with PBS, resuspended in 500 µl 1X binding buffer and incubated in 5 µl Annexin V-FITC and 5 µl PI solution in the dark for 15 min at RT. Cells were analyzed using an integrated BD Accuri™ C6 system (BD Biosciences). The gates for flow cytometry was set using following steps: Target cell population was circled by detecting unstained cells. Annexin V-FITC single positive cells were detected, following which fluorescence compensation was adjusted to ensure no particles were in the upper left (UL) and upper right (UR) quadrants. PI single positive cells were detected and fluorescence compensation was adjusted to ensure no particles were present in the upper right (UR) and lower right (LR) quadrants. A total of four populations of cells were observed: i) Cells that were viable and not undergoing apoptosis are both FITC-Annexin V and PI-negative (LL); ii) cells undergoing apoptosis were FITC-Annexin V-positive and PI-negative (LR); iii) the cells that are undergoing late apoptosis or necrotic are both positive for FITC-Annexin V and PI (UL and UR).
Annexin 5 fitc and pi staining kit
Manufactured by BD
Sourced in United States
The Annexin V-FITC and PI staining kit is a laboratory tool used for the detection and quantification of apoptosis in cells. Annexin V, a calcium-dependent phospholipid-binding protein, is conjugated to the fluorescent dye FITC (fluorescein isothiocyanate) to identify cells undergoing apoptosis. Propidium iodide (PI) is included to identify cells with compromised cell membranes, which can occur during late apoptosis or necrosis.
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Lab products found in correlation
Accuri c6 system, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
α smooth muscle actin α sma, by Cell Signaling Technology (1 mentions)
Tmrm assay kit, by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Gapdh 60004 1 ig, by Proteintech (1 mentions)
Cell counting kit 8 (cck8), by Apexbio (1 mentions)
Aurora 10, by Cytek (1 mentions)
5 protocols using annexin 5 fitc and pi staining kit
1
Annexin V-FITC and PI Assay for Cell Apoptosis
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MTT and Annexin V-FITC Cytotoxicity Assay
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Drug sensitivity assay was performed by MTT test and apoptosis was quantified by Annexin V-FITC and PI staining kit from BD PharmingenTM (San Jose, CA).
3
Cell Cycle and Apoptosis Analysis
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Cell cycle was determined by using propidium iodide (PI) DNA staining. Briefly, 2 × 106 cells were harvested and fixed in 70% (v/v) cold ethanol for 1 h. After washing 3 times with cold PBS, the supernatant was removed, and cells were resuspended in 1 mL of DNA staining solution (PBS containing 2% PI (wt/v) and 2% DNase free RNase (wt/v) for 30 min at room temperature and in the dark. Cells were then analyzed by FACS Canto II (BD, USA) using 488-nm laser for excitation. Red fluorescence of PI (> 600 nm) was measured with side scatter (SSC) and forward scatter (FSC), collecting at least 20,000 events. Residual debris was gated-out by using multilabel microbeads with sizes of 2 and 3 µm.
Apoptotic cells are detected by Annexin V-FITC and PI staining kit (BD, USA). Single and co-culture cells were collected and stained with Annexin V-FITC for 10 min, then with PI for 5 min at room temperature, in the dark. Stained cells were measured on FACS Canto II and data was analyzed by FACS Diva software. The results were presented as percentage of late and early apoptotic cells.
Apoptotic cells are detected by Annexin V-FITC and PI staining kit (BD, USA). Single and co-culture cells were collected and stained with Annexin V-FITC for 10 min, then with PI for 5 min at room temperature, in the dark. Stained cells were measured on FACS Canto II and data was analyzed by FACS Diva software. The results were presented as percentage of late and early apoptotic cells.
4
Mefunidone Induced Kidney Injury Amelioration
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Mefunidone (Lot No. 21062601) was synthesized by the Pharmaceutical Sciences Department, Central South University (Changsha, China). Folic acid (FA) (F8758) and sodium bicarbonate (V900182) were purchased from Sigma-Aldrich. Antibodies against proteins NGAL (ab216462) and collagen Ⅰ (ab270993) were purchased from Abcam. Antibodies against Bax (#2772), cleaved-caspase3 (#9664), and α-smooth muscle actin (α-SMA) (#19245) were purchased from Cell Signaling Technology. Antibodies against proteins bcl2 (#T40056) were purchased from Abcam. Antibodies against OxPhos (45–8,099) were purchased from Invitrogen. Antibodies against E-cadherin (20874-1-AP), vimentin (60330-1-Ig), α-tubulin (66031-1-Ig), and GAPDH (#60004-1-Ig) were purchased from Proteintech. A tissue reactive oxygen species assay kit (DHE) (#D7008) was purchased from Sigma-Aldrich. FITC Annexin V and PI staining kit (556547) was purchased from BD Pharmingen. Cell counting kit-8 (K1018) was purchased from APExBIO Technology LLC. The TMRM assay kit (T668) was purchased from Thermo Fisher Scientific. Blood urea nitrogen (BUN) (C013-2-1) and serum creatinine (SCr) (C011-2-1) kits were obtained from the Nanjing Jiancheng Bioengineering Institute. All the chemicals used in this study were of analytical grade.
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Apoptosis Analysis of BLM-Induced Cell Death
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MLE-12 cells were inoculated in each well of 6-well plates with DMEM/F12 medium containing 10% fetal bovine serum (FBS) for 24 h. Each plate contained three groups: control, BLM, and BLM plus MFD group. After incubation in medium with 2% FBS for 12 h, MFD group was pretreated with MFD (40 μg/ml) for 2 h. Afterwards, BLM group and MFD group were incubated with BLM (400 μg/ml) for 24 h to induce cell death. Cells were then collected, and stained with the FITC Annexin V and PI staining kit (BD Pharmingen, USA) according to the manufacturer’s instructions. This assay discriminates between intact (FITC−/PI−), early apoptotic (FITC+/PI−), and later apoptotic (FITC+/PI+) cells. Data were obtained with an Aurora-10 (Cytek) flow cytometer and analyzed with FlowJo 10.1.
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