Cell apoptosis was detected using an Annexin V-FITC/propidium iodide (PI) apoptosis assay kit (#556547, BD Pharmingen, Lake Franklin, NJ, USA). HUVECs or HBMECs were cultured in 12-well plates and harvested after OGD/R treatment. The cells were then stained with Annexin V-FITC and PI according to the manufacturer’s instructions and analyzed using flow cytometry. The total apoptosis rate (%) was calculated as (Annexin V+ PI− cells + Annexin V+ PI+ cells)/total number of cells × 100. The early apoptosis rate (%) was calculated as Annexin V+ PI− cells /total number of cells × 100. The late apoptosis rate (%) was calculated as Annexin V+ PI+ cells /total number of cells × 100.
Annexin 5 fitc propidium iodide apoptosis assay kit
Manufactured by BD
Sourced in United States
The Annexin V-FITC/propidium iodide (PI) apoptosis assay kit is a laboratory tool used to detect and quantify apoptosis, a form of programmed cell death. The kit contains Annexin V conjugated with the fluorescent dye FITC and the nuclear stain propidium iodide. These components allow for the identification of cells undergoing early and late stages of apoptosis.
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5 protocols using annexin 5 fitc propidium iodide apoptosis assay kit
1
Quantifying Cell Apoptosis by Annexin V-FITC/PI
2
Annexin V-FITC/PI Apoptosis Assay
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Apoptosis was detected using the Annexin V-FITC/propidium iodide (PI) apoptosis assay kit (BD Biosciences, Singapore). Briefly, cells were washed with cold PBS and resuspended in 100 μL of 1× Annexin V binding buffer containing 5 μL of Annexin V-FITC and 5 μL of PI. After incubation for 15 min, 400 μL of 1 × Annexin V binding buffer were added to the cells. The percentage of apoptotic cells was analyzed using a flow cytometer (BD Accuri C6: BD Biosciences, San Jose, CA).
3
Apoptosis and ROS Analysis by Flow Cytometry
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Annexin V–FITC/propidium iodide (PI) apoptosis assay kit (BD, Franklin Lake, NJ, USA) was used to analyze the apoptosis rate by flow cytometry. In brief, cells were grown to about 70% confluence in six‐well plates and treated with 30 mm HG and/or 0.5 mm Pyr for 72 h. Cells in each sample were washed twice with PBS and then resuspended in 1× Binding Buffer. Thereafter, the suspension (1 × 105 cells) was transferred to a 5‐mL culture tube, and Annexin V–FITC and PI were added to each culture tube. Finally, cells were measured by a flow cytometer (BD). Besides, ROS levels were detected by a fluorometric assay, using the 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA; Sigma Aldrich, St. Louis, MO, USA) as a fluorescence probe [18 ]. Sodium pyruvate (Pyr) was purchased from Sigma Aldrich.
4
Apoptosis Assay for PC3 and HaCaT Cells
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The PC3 and HaCaT cells were cultured and harvested under the conditions
mentioned in the Cell Culture section and seeded in 12-well plates
(1 × 105 cells per well). After 24 h of preincubation,
the cells were treated with the tested compounds at IC50 concentrations and incubated for 72 h. The apoptotic effect was
performed using the annexin V-FITC/propidium iodide (PI) apoptosis
assay kit (Becton Dickinson, Pharmingen) according to the manufacturer’s
instructions and analyzed by flow cytometry (Becton Dickinson). The
cells which were annexin V-FITC-positive and PI-negative were identified
as early apoptotic and both annexin V-FITC- and PI-positive as late
apoptotic or necrotic. The experiment was repeated three times.
mentioned in the Cell Culture section and seeded in 12-well plates
(1 × 105 cells per well). After 24 h of preincubation,
the cells were treated with the tested compounds at IC50 concentrations and incubated for 72 h. The apoptotic effect was
performed using the annexin V-FITC/propidium iodide (PI) apoptosis
assay kit (Becton Dickinson, Pharmingen) according to the manufacturer’s
instructions and analyzed by flow cytometry (Becton Dickinson). The
cells which were annexin V-FITC-positive and PI-negative were identified
as early apoptotic and both annexin V-FITC- and PI-positive as late
apoptotic or necrotic. The experiment was repeated three times.
5
Toosendanin-Induced Apoptosis in Cancer Cells
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Purified toosendanin (source: root bark and bark of Melia toosendan; molecular formula (MF): C30H38O11; molecular weight (MW): 574.62; purity ≥98%) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). RPMI-1640 medium, phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), Cell Counting Kit-8 (CCK-8) and horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (dilution 1:2,500; cat. no. HS101) were purchased from TransGen Biotech, Inc. (Beijing, China). FBS was obtained from HyClone Laboratories (GE Healthcare, Chicago, IL, USA), while the Annexin V-FITC/Propidium Iodide (PI) Apoptosis Assay kit was obtained from Becton Dickinson (BD Biosciences, San Jose, CA, USA). The Hoechst 33258 staining kit was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Antibodies against B-cell lymphoma 2 (Bcl-2; dilution 1:2,000; cat. no. ab182858), Bcl-2-associated X protein (Bax; dilution 1:1,000; cat. no. ab32503), cytochrome c (dilution 1:5,000; cat. no. ab133504), caspase-3 (dilution 1:5,000; cat. no. ab32351), caspase-8 (dilution 1:1,000; cat. no. ab108333), caspase-9 (dilution 1:1,000; cat. no. ab32539), poly(ADP-ribose) polymerase (PARP; dilution 1:1,000; cat. no. ab32138) and GAPDH (dilution 1:2,500; cat. no. ab9485) were purchased from Abcam (Cambridge, UK).
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