Rat skin fibroblasts FR (ATCC® CRL1213™) were seeded in 96-well plates at 1 × 105 cells/mL and allowed to reach log phase growth. On day four, complete growth media was aspirated. In initial studies, 100 μl of one of the following treatments were added: 0, 0.00002, 0.0002, 0.002, 0.02, 0.2, 2, 20, 200 or 500 μM concentrations of DON (Sigma-Aldrich) or the positive control 6 μM camptothecin (Sigma-Aldrich) in OptiMem (Invitrogen) with 3% FBS. Following a one, two, four or six-hour incubation, the treatments were aspirated and the cells were assessed for differences in apoptosis and cell viability. For later studies, 100 μl of the following treatments were added: 0, 2, 20, or 200 μM concentrations of DON (Sigma-Aldrich) and the positive control 6 μM camptothecin (Sigma-Aldrich) in OptiMem (Invitrogen) 3% FBS. Following forty-eight hours incubation, the treatments were aspirated and the cells were assessed for differences in apoptosis and cell viability. The Annexin V FITC assay kit (Cayman Chemical 600300) was used to detect apoptosis and cell viability. Absorbance and fluorescence were read at 485nm excitation/535nm emission. Results were obtained using a Multimode Detector DTX 880 (Beckman Coulter).
Annexin 5 fitc assay kit
Manufactured by Cayman Chemical
Sourced in United States
The Annexin V FITC Assay Kit is a laboratory product that is used to detect and quantify apoptotic cells. It contains Annexin V, a protein that binds to phosphatidylserine, a molecule that is exposed on the surface of cells undergoing apoptosis. The Annexin V is labeled with the fluorescent dye FITC, allowing the detection of apoptotic cells by flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Lectin pna, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Camptothecin, by Merck Group (1 mentions)
Dtx880, by Beckman Coulter (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Caspase glo assay kit, by Promega (1 mentions)
Victor3 plate reader, by PerkinElmer (1 mentions)
Genios plate reader, by Tecan (1 mentions)
Spectramax gemini em fluorescence microplate reader, by Molecular Devices (1 mentions)
Sulforaphane, by Cayman Chemical (1 mentions)
Facs tube, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Softmax pro 5, by Molecular Devices (1 mentions)
Spectra max m2 luminometer, by Molecular Devices (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Ecm gel, by Merck Group (1 mentions)
Collagen 4, by Merck Group (1 mentions)
26 protocols using annexin 5 fitc assay kit
1
Evaluating Cytotoxicity and Apoptosis in Rat Fibroblasts
2
Apoptosis Induction and Analysis
3
Apoptosis and Cell Death Analysis
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Early-stage apoptotic cells and dead cells were analyzed using an Annexin V FITC Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Early-stage apoptotic cells stained using Annexin V FITC were detected with excitation and emission wavelengths of 560 nm and 595 nm, respectively, whereas dead cells stained using PI were detected with excitation and emission wavelengths of 485 nm and 535 nm, respectively, using a SpectraMAX Gemini EM Fluorescence Microplate Reader (Molecular Devices, San Jose, CA, USA).
4
Sulforaphane-Induced Apoptosis Analysis
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Sulforaphane (Cas No: 4478-93-7), Annexin V FITC assay kit and JC-1 were purchased from Cayman Chemical Company (Cayman Chemical, Michigan, Ann Arbor, USA). Lectin PNA and H2DFCDA were purchased from Thermo Fisher. All other chemicals used in the study were purchased from Sigma (Sigma, Aldrich Chemical Company, Burlington, Massachusetts, USA).
5
Annexin V Assay for Apoptosis
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Exposed phosphatidyl serine was measured using an Annexin V FITC Assay kit (Cayman, Ann Arbor, MI, USA) following the manufacturer’s indications. Briefly, 2.5 million NEU/mL were placed into FACS tubes (FALCON; Becton Dickinson, Franklin Lakes, NJ, USA) in the presence of 40 µL of APTP, HHC, or HLT sera or 10 µL of zymosan (50 µg/mL) for 8 hours. A total of 200 µL of binding buffer was then added, and the tubes were centrifuged to remove the supernatants. The cell pellets were resuspended in 50 µL of staining solution (FITC-Annexin V-propidium iodide) for 10 minutes. Next, the cells were analyzed using a Facscalibur flow cytometer (Becton Dickinson) and the data were processed with the FlowJo software for early apoptosis and late apoptosis.
6
Quantifying Apoptosis with Annexin V Staining
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Annexin V staining of cells was done with Annexin V FITC Assay kit (Cayman Chemicals, Ann Arbor, MI, USA), according to the manufacturer's instructions. Samples were acquired on BD FACS Calibur Flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using Flowjo version 9.3.3 software (Tree Star Inc., Ashland, OR, USA).
7
Assessing Cell Damage and Toxicity
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To assess possible cell damage and toxicity effects of the used compounds, we used the commercial QuantiChrom Lactate Dehydrogenase Kit (BioAssays Systems, Hayward, CA, USA) for a colorimetric kinetic determination of lactate dehydrogenase (LDH) activity. The analysis was performed according to the manufacturer’s instructions. The culture media from PBMCs, stimulated as indicated in the figure legends, were assessed for released LDH, with a detection limit from 2 IU/L up to 200 IU/L. Cell apoptosis was assessed by means of an annexin V FITC assay kit (Cayman Chemical) via FACS analysis gated on the lymphocytes. Propidium iodide served as a marker of cell death in this assay.
8
Annexin V-FITC Assay for Detecting Apoptotic Cells
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Binding of annexin V to translocate the phospholipid phosphotidylserine (PS) allows for the detection and analysis of apoptotic cells
[51 (link),56 (link),57 (link)]. These assays were performed using the Annexin V FITC Assay Kit as indicated by the manufacturer (Cayman Chemical Co.) with a few modifications. Briefly, C6/36 clonal cell lines stably expressing αDENV-U143-FL, αDENV-ΔU143-ΔN Bax or αDENV-U143-ΔN Bax ΔN Bax and wild type C6/36 cells were infected with each DENV serotype (MOI = 0.1). At 48 hpi 1 × 106 clonal cells were scraped and placed in a well of a 96 well black opaque microtiter plate in triplicate for each clonal cell type assayed. FITC-annexin V microtiter plates were assayed for FITC-annexin V binding at 485 nm with the Spectra max M2 luminometer (Molecular Devices) and analyzed with Softmax Pro 5.4.5. Uninfected clonal and wild type C6/36 cells were also assayed as an additional negative control. Assays were performed in triplicate. Error bars indicate standard deviation of three independent experiments.
[51 (link),56 (link),57 (link)]. These assays were performed using the Annexin V FITC Assay Kit as indicated by the manufacturer (Cayman Chemical Co.) with a few modifications. Briefly, C6/36 clonal cell lines stably expressing αDENV-U143-FL, αDENV-ΔU143-ΔN Bax or αDENV-U143-ΔN Bax ΔN Bax and wild type C6/36 cells were infected with each DENV serotype (MOI = 0.1). At 48 hpi 1 × 106 clonal cells were scraped and placed in a well of a 96 well black opaque microtiter plate in triplicate for each clonal cell type assayed. FITC-annexin V microtiter plates were assayed for FITC-annexin V binding at 485 nm with the Spectra max M2 luminometer (Molecular Devices) and analyzed with Softmax Pro 5.4.5. Uninfected clonal and wild type C6/36 cells were also assayed as an additional negative control. Assays were performed in triplicate. Error bars indicate standard deviation of three independent experiments.
9
Anticancer Potential of Cell Lines
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The cell lines MCF-7, MDA-MB-231, MDA-MB-453, ZR-75-1, HT-29, A549 and HEK293 were purchased from NCCS Pune, India. MEF was a gift from Dr. Sougata Saha, Tezpur University, India. DMEM (Dulbecco’s Modified Eagle Medium) and FBS (Fetal Bovine Serum) were purchased from Life Technologies, USA. MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide), Collagen IV, ECM gel and Doxorubicin were also purchased from Sigma Aldrich and Mitomycin C was purchased from HiMedia, India. Annexin-V-FLUOS Staining Kit purchased from Roche, USA and Annexin V FITC Assay Kit was purchased from Cayman, USA. Antibodies used in this study were procured from Cell Signalling Technology, USA.
10
Annexin V-FITC/PI Apoptosis Assay
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Cells transfected with ATGL and BNIP3 ΔTM plasmids were seeded in a 12-well plate. After 48 h of over-expression, the cells were collected and stained with an Annexin V-FITC/propidium iodide (PI) following manufacturer’s protocol of Annexin V FITC Assay kit (Cayman Chemical, cat. Number 600300). After an incubation for 10 min at room temperature in the dark, the apoptotic rate of the cells was detected by flow cytometry (CytoFLEX S, Beckman Coulter, USA). CytExpert software was used to analyze the results.
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