The percentage of necrotic/apoptotic cells was investigated by a flow cytometer. Following ZnPc-PDT, the SW480 cells were resuspended and stained by an ApoFlowEx® FITC Kit (EXBIO, Vestec, Czech Republic). Then, 5 μL of annexin-V-FITC, 5 μL of PI, and 190 μL of binding buffer were added to each sample. After 10 min at room temperature in a dark room, the percentage of apoptotic/necrotic cells was studied by flow cytometry.
Apoflowex fitc kit
Manufactured by Exbio
Sourced in Czechia
The ApoFlowEx® FITC Kit is a laboratory equipment product designed for the detection and analysis of apoptosis. It utilizes Annexin V conjugated with the fluorescent dye FITC to identify and quantify apoptotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Macsquant, by Miltenyi Biotec (2 mentions)
Casy tt, by Roche (2 mentions)
Macsquant 10 flow cytometer, by Miltenyi Biotec (1 mentions)
Cyflow space flow cytometer, by Sysmex (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Cytoflex cytometer, by Beckman Coulter (1 mentions)
Dialysis membrane, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Px 12, by Merck Group (1 mentions)
Depc water, by Merck Group (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Tnf α elisa kit, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Retinoic acid, by Merck Group (1 mentions)
Belinostat pxd101, by Selleck Chemicals (1 mentions)
Idarubicin, by Merck Group (1 mentions)
3 deazaneplanocin a, by Cayman Chemical (1 mentions)
Guava easycyte 8ht flow cytometer, by Merck Group (1 mentions)
Iron 3 acetylacetonate, by Merck Group (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
20 protocols using apoflowex fitc kit
1
Quantifying Cell Death via Flow Cytometry
2
Evaluation of Cytotoxicity in THP-1 Cells
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Example 34
The THP-1 cell line was cultured in a medium with the addition of 10% fetal bovine serum. After achieving a sufficient density and viability (measured by means of an automatic cell calculator CASY TT, Roche), the cells were seeded into a 6-well panel in 2 ml of 10% medium. The tested film was added to the cells in an amount of 1 and 0.5 mg/ml. After 24 and 72 hours of incubation, the cells were washed and their viability and the occurrence of cell death were detected by means of the detection kit ApoFlowEx® FITC Kit (Exbio) on a flow cytometer MACSQuant® (Miltenyi Biotec). The cells were evaluated as viable in case no propidium iodide fluorescence was detected.
3
Cell Viability Assay of Test Film
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Example 35
The THP-1 cell line was cultured in a medium with the addition of 10% fetal bovine serum. After achieving a sufficient density and viability (measured by means of an automatic cell calculator CASY TT, Roche), the cells were seeded into a six-well panel in 2 ml of 10% medium. The tested film was added to the cells in an amount of 1 and 0.5 mg/ml. After 24 and 72 hours of incubation, the cells were washed and their viability and the occurrence of cell death were detected by means of the detection kit ApoFlowEx® FITC Kit (Exbio) on the flow cytometer MACSQuant® (Miltenyi Biotec). The evaluation of the presence of cell death (apoptosis and necrosis) was conducted according to the recommendation of the kit producer. In brief: the population of the individual cells was divided based on the fluorescence intensity of propidium iodide and Annexin V-FITC into 3 groups: negative in both channels (living cells), positive just in the channel for Annexin V-FITC (apoptotic cells) and positive cells for the channel propidium iodide+/−Annexin V-FITC (necrotic cells).
4
Apoptosis Analysis of Oxaliplatin-Treated SW480/Res Cells
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Annexin V/PI assay was applied to examine the impact of Nrf2 siRNA on the oxaliplatin-induced apoptosis in SW480/Res cells. First, SW480/Res cells were seeded in a six-well plate (2×105 cells per well). Then, the cells were transfected with Nrf2 siRNA and treated with oxaliplatin for 48 hr. The cells were harvested using trypsin EDTA 0.05%. Subsequently, the cells were washed twice with PBS, and stained by applying ApoFlowEx® FITC Kit (EXBIO Praha, a.s., Czech Republic). Finally, the apoptotic cells were analyzed through MACSQuant® 10 Flow Cytometer (Miltenyi Biotec, Germany). FlowJo (7.6.1) software was used for analysis of raw data 21 (link),22 (link).
5
Apoptosis Detection by Annexin V Assay
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To investigate the effect of the aforementioned compounds on programmed cell death, the appearance of anionic phosphatidylserine (PS) on the surface of the cells was assayed by the Apo FlowEx ® FITC kit (ExBio, ED7044). In brief, cells (10 × 10 4 cells/well) were seeded into 12-well cell culture plates and exposed with elected concentrations of the compounds for 48 h. Then, the cells were collected, washed with phosphate-buffered saline, and incubated (15 min) with binding buffer, propidium iodide (PI), and Annexin V according to the manufacturer's instructions. [ 21 , 22 ] Using the CyFlow ® Space flow cytometer (Sysmex Partec), the percentage of apoptotic cells (Annexin V + /PI -and Annexin V + /PI + cells) was detected. It should be noted that unlabeled cells were applied to remove autofluorescence and all trials were repeated three times.
6
Apoptosis and Necrosis Profiling Using ApoFlowEx FITC Kit
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The ApoFlowEx FITC Kit was used to identify the early and late phases of apoptosis, as well as the necrosis profiles of the treated cells (Exbio, Czech Republic). Briefly, treated cells were rinsed (× 3) with PBS, trypsinized, and then washed (× 3) with 500 µL of binding buffer (BB) [Tris–HCl (1 m, pH 8.0), EDTA (500 mm), and NaCl (5 m)]36 (link),37 (link). After draining the supernatant, the cells were resuspended in 100 µL of BB and 5 µL of annexin V-FITC. After 15 min of incubation at 24 ± 2 °C in dim light, the cells were washed once more with BB. Following supernatant removal, 100 μL of the BB and 5 μL of PI staining solution were added and incubated for additional 5 min at 24 ± 2 °C in the dark. The cells were examined using a BD FACSCaliburTM flow cytometer (FACSQuant; Milteny, Germany) equipped with FITC (green) emission filters of 515–545 nm and PI emission filters of 600 nm (red). Each experiment recorded a total of 10,000 occurrences.
7
Apoptosis and Necrosis Quantification in HCT116 and SW480 Cells
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The percentage of apoptosis and necrosis of HCT116 and SW480 cells in miR-330 and mock group were determined by annexinV/PI assay. Briefly, 2×10 5 cells seeded per well of 6-well cell culture plate. After 48 h the cells detached via trypsin EDTA 0/05% and stained with ApoFlowEx ® FITC Kit; was bought from EXBIO (EXBIO, Vestec, Czech Republic) according to the EXBIO instructions. The percentage of apoptotic cells were assessed through MACS Quant 10 flow cytometry instrument (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany). For analysis, the cells were gated from FCS and SSC
8
Assessing Cell Viability and Apoptosis
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The cell number and viability were assessed using the ApoFlowEx FITC Kit (Exbio, Vestec, Czech Republic) each time before seeding the cells into culture dishes and at each of the four time points. For this purpose, pooled adherent and non-adherent hAECs were counted using a Cytoflex cytometer, and then batches of 200,000–500,000 cells were suspended in 100 µL of pre-diluted buffer (Annexin V Binding Buffer 10×). The cell suspension was supplemented with 5 µL Annexin V-FITC and 5 µL propidium iodine and incubated for 15 min at room temperature, followed by analysis on a Cytoflex cytometer.
9
Cytotoxicity and Apoptosis Induction in Colon Cancer Cells
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Chloroform, ethanol, methanol, span 60, Tween 60, cholesterol, dimethyl sulfoxide (DMSO), ultrapure sodium dodecyl sulfate (SDS), and Amicon® Ultra Centrifugal Filter Devices (Amicon Ultra-15 Membrane MWCO 30 KDa) were purchased from Merck company (Germany). Phosphate buffered saline tablet (PBS) provided by Bio Basic Company(Canada). The PX-12 (PubChem CID: 219,104), YC-1(PubChem CID: 5712), DEPC water, and dialysis membrane (MWCO 12 KDa) were obtained from Sigma Aldrich company(USA). Dulbecco's Modified Eagle's medium (DMEM), Trypsin–EDTA, and Penicillin–Streptomycin(pen-strep) were received fromBiosera company (France). Fetal bovine serum (FBS) was bought from Gibcocompany (USA)0.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Atocelcompany(Australia).ApoFlowEx FITC Kit (Annexin V/PI) was acquired from EXBIO company (Czech Republic). The flow cytometer used in this study is a FACScalibur belonging to BD Biosciencescompany (Canada). Sybr Green master mix real-time PCR was purchased from Ampliqoncompany (Denmark). Intestinal cancer cells (HT-29) and human foreskin fibroblast (HFF) were supplied from the National Center for Genetic and Biological Resources of Iran.
10
Staphylococcus haemolyticus Apoptosis Induction
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To analyze the ability of S. haemolyticus to induce apoptosis in the PHSF cells, adherent PHSF cells were infected with washed S. haemolyticus. After 24 h, cells were de-attached by trypsin and apoptosis was detected by flow cytometry using Annexin V/PI staining (ApoFlowEx FITC Kit, exbio). Early apoptotic cells were stained with annexin V alone, whereas necrotic cells and late apoptotic cells stained with both annexin V and propidium iodide.
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