After treatment with iso-suillin for 48 h, the cells were double stained with an Annexin V-FITC/PI Kit (MultiSciences Biotech, China) according to the manufacturer's instructions. The apoptosis rate and cell cycle distribution were analyzed using flow cytometry (Epics-XL II, Beckman Coulter, USA).
Annexin 5 fitc pi kit
Manufactured by MultiSciences Biotech
Sourced in China
The Annexin V-FITC/PI kit is a laboratory reagent used to detect and quantify apoptosis, or programmed cell death, in cell samples. It contains Annexin V, a protein that binds to phosphatidylserine residues exposed on the surface of apoptotic cells, and propidium iodide (PI), a dye that stains the DNA of cells with compromised cell membranes. The binding of Annexin V-FITC and the uptake of PI can be detected and analyzed using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Fc500 mcl machine, by Beckman Coulter (2 mentions)
Epics xl 2, by Beckman Coulter (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Accuri c6, by BD (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Flow cytometry, by Beckman Coulter (1 mentions)
8 protocols using annexin 5 fitc pi kit
1
Apoptosis and Cell Cycle Analysis
2
Apoptosis Quantification in SKOV3 Cells
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SKOV3 cells of different groups (control: Cells without any treatment, lncRNA-ATB-NC and lncRNA-ATB-shRNA respectively) were harvested using 0.2% trypsin, washed with phosphate buffered saline (PBS) and then fixed with 70% ethanol overnight at 4°C. The number of apoptotic cells was quantified using the AnnexinV-FITC/PI kit (cat no. 70-AP101-100; MultiSciences) according to the manufacturer's instructions. Cell apoptosis rate was measured using a FACS Calibur flow cytometer (BD Biosciences) and the data were analyzed using FlowJo software (version 7.6.1; FlowJo LLC). The assay was performed in triplicate.
3
Quantifying Chondrocyte Apoptosis via Flow Cytometry
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An Annexin V-FITC/PI kit (MultiSciences Biotech Co., Ltd., Hangzhou, China) was used to assess rates of chondrocyte apoptosis based on provided directions. Briefly, chondrocytes were spun down for 5 min at 1000 rpm, washed twice using cold PBS, and resuspended in 500 µl of binding buffer containing 5 µl each of PI and Annexin V-FITC. Following a 15 min incubation protected from light, cells were assessed using a FACScan flow cytometer (Becton-Dickinson, USA) in order to quantify apoptotic rates.
4
Annexin V Apoptosis Assay
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The Annexin V-FITC/PI kit (Multiscience) was used to analyze the extent of apoptosis. Briefly, cells were collected by trypsinization and washed three times with phosphate-buffered saline (PBS), then resuspended in 500 µl binding buffer with 5 µl Annexin V-FITC and 10 µl PI. Cells were incubated for 5 min in the dark at room temperature. The cells were then analyzed using a FC500 MCL machine (Beckman Coulter) at 10,000 events/sample.
5
Secretome study of alveolar epithelial A549 cells
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As human alveolar epithelial carcinoma A549 cells (CCL-185, ATCC) are very tolerant to SFM, we chose them as a cell model for our secretome study [15 (link)]. Firstly, A549 cells were maintained in phenol red-free Dulbecco’s Modified Eagle Medium Nutrient Mixture (DMEM/F12) (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37°C in a humidified atmosphere containing 5% CO2. When A549 cells were grown to approximately 60-70% confluence, they were washed five times with SFM to remove albumin and other elements contained in FBS. Cells were then either infected with 10 CFU/cell of M. pneumoniae in SFM or left untreated for further conditioned media (CM) collection. Cell viability in SFM was assessed by MTT test and trypan blue exclusion assay, and the cell death was assessed by apoptosis assay using the Annexin V-FITC/PI Kit (Multiscience, Hangzhou, China).
6
Annexin V-FITC/PI Apoptosis Assay
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Apoptosis was assessed using an Annexin V-FITC/PI kit (Multi Sciences, AP101, China). KGN cells from each group were seeded in 6-well plates at a density of 1 × 10 5 cells per well. After treatment, the culture medium was discarded and the cells were washed with phosphate-buffered saline (PBS) before digestion with 0.25% trypsin-EDTA (Gibco, USA). Digestion was terminated in a medium containing 10% FBS, followed by centrifugation (1000 rpm, 5 min). A 1× working solution of 5× Binding Buffer was prepared and the cells were suspended in 500 µL of this working solution. Annexin V-FITC (5 µL) and PI (10 µL) were added to each tube, mixed, and incubated for 5 min at room temperature. Flow cytometric analysis was conducted using a BD Accuri™ C6 (Becton Dickinson, USA) cytometer.
7
Annexin V-FITC Apoptosis Assay
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After transfection, cells (1 × 106 cells/mL) were collected and digested with 0.25% trypsin. Based on the manufacturer's instructions, apoptosis of MIN6 cells was determined with an Annexin V-FITC/PI kit (Multi Sciences, China). Finally, apoptosis was measured by flow cytometry (Beckman, USA).
8
Annexin V-FITC/PI Apoptosis Assay
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The Annexin V-FITC/PI kit (Multiscience) was used to analyze the extent of apoptosis. Briefly, cells were collected by trypsinization, washed three times with PBS, and then resuspended in 500 μL binding buffer with 5 mL Annexin V-FITC and 10 μL PI. Cells were incubated for 5 min in the dark at room temperature. The cells were then analyzed using a FC500 MCL machine (Beckman Coulter) at 10,000 events/sample.
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