The apoptosis percentage was detected by flow cytometry using Annexin V-FITC/PI (BioLegend, USA) staining. The control, METH, and METH+MCC950 groups of IEC-6 cells were washed twice with PBS and centrifuged at approximately 1000×g at 4°C for 5 min. After being resuspended in 100 μl of binding buffer, the cells were placed on ice with 5 μl of Annexin V-FITC for 15 min. Finally, 1 μl of Propidine Iodide (PI) was added to the cells at ambient temperature for 2 min. A flow cytometer was used to assess the percentage of apoptotic cells.
Annexin 5 fitc pi
Manufactured by BioLegend
Sourced in United States
Annexin V-FITC/PI is a fluorescent staining reagent used in flow cytometry to detect and quantify apoptosis. Annexin V binds to phosphatidylserine, which is exposed on the cell surface during early apoptosis. Propidium iodide (PI) is used to detect late stage apoptosis and necrosis by staining DNA in cells with compromised cell membranes.
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Lab products found in correlation
Accuri c6, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Apc conjugated anti mouse f4 80, by BioLegend (1 mentions)
Pe conjugated anti human cd163, by BioLegend (1 mentions)
Pe conjugated anti mouse cd16 32, by BioLegend (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Facscalibur, by BD (1 mentions)
Ckx41, by Olympus (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BioLegend (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Bio-Rad (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Dcfh da, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
9 protocols using annexin 5 fitc pi
1
Apoptosis Analysis by Flow Cytometry
2
Apoptosis Profiling of Adipose-Derived Stem Cells
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Cellular apoptosis assays were performed after 14 days of cell proliferation by Annexin V-FITC/PI from BioLegend (San Diego, Calif.). Cells were collected from each well by trypsin treatment. After wash, cells were resuspended in 17 μl of staining buffer with 1 μL of FITC-Annexin V and 2 μL of propidium iodide solution. The cells were then incubated for 15 minutes at room temperature in the dark. After incubation, flow cytometry analysis was performed using 488 nm excitation to measure green fluorescence emission for Annexin V (ie, 530/30 bandpass/FL-1) and red fluorescence emission for propidium iodide (ie, 610/20 bandpass/FL-3). For the positive controls, ADSC cells were cultured in Dulbecco's Modified Eagle Medium/10% fetal bovine serum with ascorbic acid at 10 μg/ml until 70–80% confluency. UVC (254 nm/50 J/m2) was used to stimulate apoptosis in cultured ADSC with a Stratalinker 1,800 UV crosslinker (Stratagene, La Jolla, Calif.) and then incubated for 24 and 48 hours. All cells were collected for Annexin/PI staining, followed by Fluorescence-activated cell sorting analysis.
3
Quantitative Apoptosis Assay in Caco-2 Cells
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Annexin V-FITC/PI double staining (BioLegend, San Diego, CA, USA) was used to quantitatively assay apoptotic Caco-2 cells. According to the detection kit protocol, Caco-2 cells were seeded at 3 × 105 density per well onto sterile 12-well plates. After a 24 h culture, 0, 15, 30, and 60 μM of RT2 were added and continually incubated for 24 h. Cells were then trypsinized and washed with ice-cold phosphate-buffered saline (PBS) twice. The detached cells were suspended in Annexin V binding buffer and stained with Annexin V-FITC/PI (BioLegend, San Diego, CA, USA). After gently mixing, the cells were then incubated for 15 min at room temperature in the dark. The stained cells were then immediately processed using a BD FACSCanto II Flow Cytometer (BD Biosciences, San Jose, CA, USA), and the data were analyzed using BD Accuri C6 software.
4
Apoptosis Evaluation by Flow Cytometry
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Annexin V-FITC/PI (Biolegend, USA) double-stained cell apoptosis detection kit was used for fluorescent labeling, and flow cytometry (CytoFLEX, BECKMAN) was used to evaluate cell apoptosis.
5
Electroporation and Macrophage Polarization Assay
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For in vitro electroporation detection, we utilized PI (Invitrogen) staining. PI (640905, BioLegend) was added to the cell suspension simultaneously or after electroporation at 10 μg/mL. After incubation for 15 minutes, the transfection efficiencies of the samples were measured. To determine apoptosis, after electroporation, the resuspended tumor cells were stained with Annexin V-FITC/PI (640905, BioLegend), and analyzed by FC. To analyze the polarization of macrophages, the macrophages were stained with FITC-conjugated anti-human HLA-DR (11–9956-42, eBioscience, San Diego, USA), PE-conjugated anti-human CD206 (12–2069-42, eBioscience), PE-conjugated anti-human CD163 (333606, Biolegend), PE-conjugated anti-mouse CD16/32 (101307, Biolegend), and APC-conjugated anti-mouse F4/80 (123115, Biolegend) .
6
Cell Death Determination by Flow Cytometry
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Cell death types were determined using the Annexin V-FITC/PI (Biolegend, 640914) via flow cytometry of the cell lines. The cell lines (3 × 105 cells/2 mL per well) were seeded in six-well plates and then incubated for 24 h at 37 °C in 5% CO2. The cell lines were treated with the highest doses of Compounds 1–6 for 48 h. The cells were harvested from the surfaces with trypsin after incubation. PBS was used to wash the cells. Then, the cells were centrifugated at 1500 rpm for five minutes, and the cell pellets were resuspended in 1 X binding buffer. Five microlitres of fluorochrome-conjugated Annexin V and 10 μL of PI were treated with 100 μL of cell suspension and then incubated for 15 min at 37 °C in the dark. Then, 400 µL of 1 X binding buffer was added. Each sample was analysed via flow cytometry (Beckman Coulter CytoFLEX Flow Cytometer), and the data were obtained using CytExpert software. The amounts of viable, early–late apoptotic, and necrotic cells were given as a percentage of the total population. Measurements were repeated three times.
7
Apoptosis Analysis via Flow Cytometry
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After the indicated treatment, cells were collected and washed with ice-cold PBS. Cells were stained with Annexin V-FITC/PI according to the manufacturer’s instructions (BioLegend, San Diego, CA, USA). After incubation, the cells were measured by flow cytometry (FACSCalibur, BD, Franklin Lakes, NJ, USA).
8
Apoptosis and Necrosis Assay in Microglia
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To examine apoptosis and necrosis, annexin V-FITC/PI (BioLegend Inc.) assays were used. Pure microglia (1 × 106 cells/well) were seeded in six-well plates. Saline or D-TA (100 ng/ml) were treated 1 or 24 h. Then, cells were washed with PBS, and 100 µl of cold-PBS, 5 µl of FICT-annexin V, and 10 µl of propidium iodide were added in order. After 15 min at room temperature in the dark, cells were analyzed by Flow cytometer (FACSVerse, BD Biosciences). To count cell number, a 1-cm2 square was marked on bottom of plate before seeding cells then images of cells in each condition were obtained by using microscopy (CKX41, Olympus Corporation, Tokyo, Japan). The number of cells was counted by ImageJ software.
9
Macrophage Activation and Oxidative Stress
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RPMI-1640 media and FBS were purchased from Gibco, Waltham, Massachusetts, USA. Antibiotics, LPS, DCFHDA, TISO4 and H2O2 were procured from Sigma-Aldrich, St. Louis, Missouri, USA. MTT, DAPI and DABCO were purchased from SRL, India. Anti-mouse CD14-PE (Thermo-Scientific, Waltham, Massachusetts, USA), β-defensin 2 (Prospec, Hamada, Israel) and RNAiso plus (Takara, Japan) were purchased. cDNA synthesis kit and SYBR green were obtained from BIO-RAD, USA. AnnexinV-FITC/PI and CFSE were purchased from Biolegend, San Diego, USA. Anti-mouse CD14 conjugated with PE was purchased from Thermofisher, USA.
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