Cells treated with indicated exosomes or oligos were exposed to 200 μM CoCl2 for 24 h, and cell apoptosis was tested using PI/Annexin V-FITC staining (#556547, BD Biosciences, St. Louis, MO, USA) according to the manufacturer’s protocol for another 10 min. After incubation, the cells were subjected to apoptosis analysis using BD Accuri C6 Plus cytometer (Beckman Coulter, Fullerton, CA, USA), and the results were analyzed with BD Accuri C6 Plus software.
Annexin 5 fitc pi staining
Manufactured by BD
Sourced in United States
Annexin V-FITC/PI staining is a laboratory technique used to detect and quantify apoptosis, a type of programmed cell death. The kit contains Annexin V conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate) and the DNA-binding dye Propidium Iodide (PI). Annexin V binds to phosphatidylserine, which is exposed on the cell surface during apoptosis, while PI stains the DNA of cells with compromised cell membranes, typically late-stage apoptotic or necrotic cells. The dual staining allows for the differentiation of viable, early apoptotic, late apoptotic, and necrotic cell populations using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Facscan, by BD (5 mentions)
Facscalibur, by BD (3 mentions)
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Cytometric bead array kit, by BD (1 mentions)
Elx800, by Agilent Technologies (1 mentions)
Enzyme linked immunosorbent assay kit, by ABclonal (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Fluorescence activated cell sorting (facs), by BD (1 mentions)
Wizard2 link, by PerkinElmer (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Prism6 v6, by GraphPad (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Mk2206, by MedChemExpress (1 mentions)
Apoptosis detection kit, by BD (1 mentions)
Cellquest pro software, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
26 protocols using annexin 5 fitc pi staining
1
Apoptosis Analysis of Cells Treated with Exosomes or Oligos
2
Biochemical Assays for Cell Death and Inflammation
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Luciferase assays were performed using a dual‐luciferase assay kit (Cat: E1960; Promega, Madison, WI). Apoptosis was tested using PI/annexin V‐FITC staining (Cat: 556547; BD Biosciences, San Jose, CA) according to the manufacturer’s protocol. The serum AST and ALT were measured using a spectrophotometer (ELx800; BioTek, Winooski, VT). The serum IL‐6 and TNF‐α was detected using cytometric bead array kits (Cat: 51‐9000147; BD Biosciences), and IL‐1β was detected using an enzyme‐linked immunosorbent assay kit (Cat: RK00006; ABclonal, Woburn, MA).
3
Annexin V/PI Apoptosis Assay
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Cell apoptosis was analyzed using FITC-annexin V/PI staining (BD Biosciences) following the manufacturer’s protocol. Data were collected and analyzed using FlowJo (Treestar, Ashland, OR) software.
4
Apoptosis Induction by AFB1
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Induction of apoptosis was determined by FITC-Annexin V/PI staining (BD Pharmingen, San Diego, CA, USA). In total, 10,000 cells were acquired and analyzed with a flow cytometer after AFB1 exposure [24 (link),25 ].
5
Inhibition of miR-31 Induces Apoptosis
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The 21nt oligonucleotide miR-31 inhibitor (5-AGCUAUGCCAGCAUCUUGCCU-3) or negative control Scramble RNA (5-CAGUACUUUUGUGUAGUACAA-3) were transfected into HCT116 cells with or without CHIR99021 (GSK3β inhibitor). The apoptotic cells were evaluated by FITC-Annexin V/PI staining (BD PharMingen) and analyzed by FACS (Becton, Dickinson).
6
Apoptosis Quantification by Annexin V-FITC/PI
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Cell apoptosis was assessed with Annexin V-FITC/PI staining (BD Biosciences). After being washed twice with PBS, cells were harvested in binding buffer at a concentration of 1 × 106 cells/ml, and 5 μl of PI and 5 μl FITC were added to the cell suspension and incubated at room temperature in the dark for 15 min. Cell samples were analyzed via flow cytometry (FACScan, BD Biosciences), and apoptotic fractions were determined.
7
Apoptosis Quantification in MCF-7 Cells
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Apoptosis was identified by flow cytometry with Annexin V-FITC/PI staining (BD Pharmingen, San Diego, CA, USA), as described by the manufacturer’s mannuals. Briefly, MCF-7 cells were treated with each ganglioside, as described above, and then the cells were centrifuged for 5 min at 3000 rpm. To discard the cell medium, the supernatant was removed and the remaining cells were added with 100 μL of binding buffer and 3 µL of Annexin V-FITC. At the condition shielded from light, the cells were incubated at room temperature for 15 min. 100 μL of binding buffer and 3 µL of PI were added to the cells. MCF-7 cells treated with 80 µM of methanol were used as a negative control. The fluorescence of 10,000 events per sample was analyzed using a Becton- Dickinson FACScan (Becton Dickinson, Franklin Lakes, NJ, USA).
8
Thymidine Analogue Incorporation Assay
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U87, NHAs, and NSCs were seeded into a 12-well plate. After adherence,
the cells were treated for 1 h with the thymidylate synthase inhibitor
5′-fluorodeoxy-2′-uridine (FdUrd, 10 μM). The
cells were washed with DPBS and incubated for 4 and 24 h after addition
of 100 kBq [125I]ITdU. For co-incubation, EdC-NG with a
concentration of 10 mg/mL was added to U87 cells. The cell-incorporated
radioactivity was subsequently measured using the gamma counter Wizard2 (link) (PerkinElmer). To isolate the DNA of the cells,
the “DNeasy Blood & Tissue Kit” from Qiagen was
used. The procedure was done according to the “Quick-Start
Protocol” for cultured cells. After the DNA was extracted,
the samples’ radioactivities [counts/min] were measured with
the gamma counter. Subsequently the dsDNA contents [μg/mL] of
the samples were measured using a photometer. The apoptosis was visualized
by Annexin V-FITC/PI staining (BD Biosciences, Heidelberg, Germany)
and the cell viability determined using flow cytometry.
the cells were treated for 1 h with the thymidylate synthase inhibitor
5′-fluorodeoxy-2′-uridine (FdUrd, 10 μM). The
cells were washed with DPBS and incubated for 4 and 24 h after addition
of 100 kBq [125I]ITdU. For co-incubation, EdC-NG with a
concentration of 10 mg/mL was added to U87 cells. The cell-incorporated
radioactivity was subsequently measured using the gamma counter Wizard2 (link) (PerkinElmer). To isolate the DNA of the cells,
the “DNeasy Blood & Tissue Kit” from Qiagen was
used. The procedure was done according to the “Quick-Start
Protocol” for cultured cells. After the DNA was extracted,
the samples’ radioactivities [counts/min] were measured with
the gamma counter. Subsequently the dsDNA contents [μg/mL] of
the samples were measured using a photometer. The apoptosis was visualized
by Annexin V-FITC/PI staining (BD Biosciences, Heidelberg, Germany)
and the cell viability determined using flow cytometry.
9
Apoptosis Assay using Annexin V-FITC/PI
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Apoptosis assay was carried out using Annexin V-FITC/PI staining (BD Biosciences, Cat#556547) with flow cytometry. Cells were washed with cold PBS for three times and resuspended in 1 × binding buffer after 48 h treatment with Olaparib and LAE003 at IC50 concentration (Table 3 ). Cells (1 × 105) were then incubated with 5 µL annexin V-FITC and 5 µL propidium iodide (PI) for 15 min at ambient temperature (25 °C) in the dark. After the addition of 400 µL of 1 × binding buffer, the rate of cell apoptosis was measured using flow cytometer (Beckman Coulter).
10
Cell Death Assay by Annexin V-FITC/PI
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To perform the assay, 75,000 cells were seeded into 12-well plates in three replicates. The day after, culture medium was replaced by drug-containing medium. After 72 h of incubation, cell death was measured by dual Annexin V-FITC/PI staining (#556547, BD Biosciences, San Jose, CA, USA) according to manufacturer’s recommendations. For doxorubicin, whose red coloration impairs PI staining, we analyzed only AnnexinV-FITC staining.
For each sample, 10,000 cells were analyzed by flow cytometry (FACS Calibur, BD Biosciences). Data were acquired using BD CellQuestPro software. Data analysis was performed with FlowJo v10.1 (FlowJo LLC, Ashland, OR, USA) and Prism6 v6.01 (GraphPad Software Inc., La Jolla, CA, USA) software.
For each sample, 10,000 cells were analyzed by flow cytometry (FACS Calibur, BD Biosciences). Data were acquired using BD CellQuestPro software. Data analysis was performed with FlowJo v10.1 (FlowJo LLC, Ashland, OR, USA) and Prism6 v6.01 (GraphPad Software Inc., La Jolla, CA, USA) software.
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