The Dead Cell Apoptosis Kit with Annexin-V FITC and PI (V13242; Invitrogen) was used to perform cell apoptosis analysis according to the manufacturer’s instructions. Briefly, cells (3 × 106) were stained using the apoptosis kit and suspended in annexin V-binding buffer (Invitrogen). The suspended cells were used to perform flow cytometry. Cell apoptosis was determined using the FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany). In total, 10,000 cells were analyzed per measurement. Data were analyzed using the FlowJo 10.0.7 software (Treestar Inc., Ashland, OR, USA)17 (link).
Dead cell apoptosis kit with annexin 5 fitc and pi
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Macao
The Dead Cell Apoptosis Kit with Annexin V FITC and PI is a laboratory tool used for the detection and quantification of apoptotic and necrotic cells. It contains Annexin V FITC and propidium iodide (PI) to stain cells undergoing apoptosis and necrosis, respectively. The kit allows for the identification and differentiation of viable, early apoptotic, late apoptotic, and necrotic cell populations through flow cytometric analysis.
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45 protocols using dead cell apoptosis kit with annexin 5 fitc and pi
1
Annexin-V FITC and PI Apoptosis Assay
2
Cell Viability Assay Post-UVB Irradiation
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Cell viability 24 h post-UVB irradiation was determined using a Dead Cell Apoptosis Kit with Annexin V FITC and PI (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Labeled cells were analyzed by flow cytometry with a FACSCalibur (Becton Dickinson, San Jose, CA, USA). Double negative cells were considered viable. For data collection and evaluation, CellQuest software 5.2 (Becton Dickinson) and Flowjo v10.0.7 (TreeStar, Ashland, OR, USA) single-cell analysis software were used.
3
Membrane Disturbance of Neutrophils
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The disturbance of the cell membrane was monitored using Annexin V (AnV) and propidium iodide (PI). Neutrophils (1 × 106 cells/sample) were placed in an eppendorf tube, washed twice with PBS, and resuspended in solutions of FOH or FA at variable concentrations. Unstimulated cells served as a negative control, and phorbol 12-myristate 13-acetate (PMA)-treated neutrophils represented a positive control. Cells were incubated for 1 h at 37 °C, at 5% CO2, washed three times with PBS, and stained with propidium iodide and fluorescein isothiocyanate (FITC)-labeled Annexin V for 15 min, according to supplier’s instruction (Dead Cell Apoptosis Kit with Annexin V-FITC and PI, Invitrogen, Carlsbad, CA, USA). Then, cells were analyzed with flow cytometry (LSR Fortressa, BD, San Jose, CA, USA).
4
Annexin V-FITC and PI Staining
5
Cyanidin Chloride Synthesis and Biological Assays
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RPMI-1640 medium, fetal bovine serum (FBS), penicillin-streptomycin and trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA). Methylglyoxal (MG), methylthiazolyldiphenyl-tetrazolium bromide (MTT), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), L-glutathione reduced, sulfosalicylic acid (SSA), 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), NADPH, and glutathione reductase were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The EnzChek® Caspase-3 assay kits and Dead Cell Apoptosis kit with Annexin V FITC and PI were purchased from Invitrogen (Carlsbad, CA, USA). Cyanidin chloride was synthesized from quercetin according to published method [19 (link)].
6
Annexin V/PI Apoptosis Assay
7
Apoptosis Quantification with Annexin V
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Apoptotic cell population was quantified using the Dead Cell Apoptosis Kit with Annexin V FITC and PI (Invitrogen, Carlsbad, CA, USA). Cells were seeded at 50–70% confluency and left to adhere overnight. Culture media were then aspirated and replaced with complete media containing drugs at the desired concentration and incubated for 48 h at 3 °C with 5% CO2. Both adherent and floating cells were harvested, pelleted and washed in PBS before re-suspending in annexin V binding buffer. Cells were then stained with 100 μg/mL propidium iodide and 1 μL FITC annexin V for 15 min at room temperature before being assessed and analyzed by FACSAriaII flow cytometer (BD Biosciences, San Jose, CA, USA) and BD FACSDIVA software (BD Biosciences, San Jose, CA, USA), respectively.
8
Cell Proliferation and Apoptosis Assay
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The proliferation ability of cells was determined with a CCK-8 kit (DoJinDo, Tokyo, Japan). In a brief, 10 μL CCK-8 solution was added to each well of the plate and sustained for 1 h at 37 ℃. The absorbance at 450 nM was detected by a Microplate Reader (Bio-Rad) to reflect cell number. The apoptotic cells were detected by a Dead Cell Apoptosis Kit with Annexin V FITC and PI, for flow cytometry (Invitrogen). Harvested cells were suspended in Annexin binding buffer provided by the kit and stained with Annexin V-FITC and PI at room temperature for 15 min. After that, cells were subjected to the flow cytometry analysis on a MACSQuant X (Miltenyi, Bergisch Gladbach, Germany). The data was analyzed by the FlowJo software. Annexin V+/PI+ and Annexin V+/PI− cells were apoptotic cells.
9
Neutrophil Apoptosis Quantification
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Neutrophils stimulated by selected factors were stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC)-labeled Annexin V (AnV) (Dead Cell Apoptosis Kit with Annexin V-FITC and PI, Invitrogen, Carlsbad, CA, USA), according to the supplier’s instruction. The analysis of apoptosis progress was performed using flow cytometry (LSR Fortressa, BD, San Jose, CA, USA). In the second way, neutrophils were stained with the CellEvent™ Caspase-3/7 Detection Reagents kit (Invitrogen, Waltham, MA). The fluorescence was measured on a microplate fluorescence reader (H1, Biotek) - excitation: 495 nm, emission: 525 nm.
10
Apoptosis Detection by Flow Cytometry
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The Dead Cell Apoptosis Kit with Annexin V FITC and PI (Invitrogen) was used to detect apoptosis by flow cytometry. Apoptosis was induced by dsDNA transfection or cGAMP stimulation, as described above. Untreated cells served as a negative control. Harvested cells were washed in cold PBS and resuspended in 1× Annexin-binding buffer. To stain the cells, 5 μl of AlexaFluor488 Annexin-V and 1 μl of 100 μg/ml PI solution were added per 100 μl of cell suspension and incubated for 15 min at RT. Cells were analyzed by flow cytometry measuring fluorescence emission at 530 nm and >575 nm using a NovoCyte Flow Cytometer (ACEA Biosciences). Data were analyzed using FlowJo software v10.6.2 (FlowJo LLC). The gating strategy is displayed in Supplementary Fig. S8 .
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