After treatment, cells were incubated with 50 µM BrdU for 1 h. Cells were collected by tryptase and centrifugation and fixed in 70% ethanol at 4 °C for 1 h and subsequently incubated with 2 N HCl/0.5%Triton X-100 for 30 min, 0.1 M borate sodium for 2 min. After anti-BrdU-FITC (eBioscience) incubation and washing, BrdU incorporation rate were analysed by flow cytometry.
Anti brdu fitc
Manufactured by Thermo Fisher Scientific
Anti-BrdU-FITC is a fluorescently labeled antibody that binds to BrdU (bromodeoxyuridine), a synthetic nucleoside that is incorporated into the DNA of dividing cells. This product can be used to detect and quantify cell proliferation through flow cytometry or fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Bromodeoxyuridine (brdu), by Merck Group (4 mentions)
Dnase, by Thermo Fisher Scientific (2 mentions)
Bromodeoxyuridine (brdu), by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Superblock buffer, by Thermo Fisher Scientific (2 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
9 protocols using anti brdu fitc
1
BrdU Incorporation Assay for Cell Proliferation
2
Primary Mouse Embryonic Fibroblast Isolation and Characterization
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Primary MEFs isolated from E14.5 embryos were designated passage 0 (P0) and were cultured in DMEM medium (GIBCO, Life Technology) containing 15% fetal bovine serum, 100 mM L -glutamine, and penicillin–streptomycin on a gelatinized plate until they reached confluence. Equal numbers of cells (1 × 104) were plated into a well of a six-well plate. The medium was changed daily and MEFs were counted at different time points in triplicate using a hemocytometer.
For IR, etoposide and HU sensitivity assays, cells were plated on 96-well plates 24 h before irradiation or the addition of genotoxic agents (etoposide: 0.01, 0.1 and 1 μM; hydroxyurea: 20, 200 and 2,000 mM. The drug was washed away 24 h after the treatment). The relative cell density was determined 7 days after irradiation or drug treatment by fluorescence nucleotide dye CyQuant (MEFs) or by hemocytometer (v-abl transformed pre-B cells).
For cell cycle analyses, proliferating primary MEF cells were incorporated with 10 μM BrdU for 30 min and fixed and then stained with Anti-BrdU FITC (Cat# 11-5071-41, eBioscience) and propidium iodide (PI) and analysed by flow cytometry
For IR, etoposide and HU sensitivity assays, cells were plated on 96-well plates 24 h before irradiation or the addition of genotoxic agents (etoposide: 0.01, 0.1 and 1 μM; hydroxyurea: 20, 200 and 2,000 mM. The drug was washed away 24 h after the treatment). The relative cell density was determined 7 days after irradiation or drug treatment by fluorescence nucleotide dye CyQuant (MEFs) or by hemocytometer (v-abl transformed pre-B cells).
For cell cycle analyses, proliferating primary MEF cells were incorporated with 10 μM BrdU for 30 min and fixed and then stained with Anti-BrdU FITC (Cat# 11-5071-41, eBioscience) and propidium iodide (PI) and analysed by flow cytometry
3
Assessing Cell Proliferation by BrdU Incorporation
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BrdU (Sigma) was injected i.p. at 2 mg and supplemented in the drinking
water at 0.8 mg/ml from days 6–10 p.i. FLICA reagent was prepared and
added to cells according to manufacturer’s instructions (Thermofisher).
For BrdU staining, cells were permeabilized, treated with 3 μg/mL DNAse
(Invitrogen) and subjected to intracellular staining with either anti-BrdU-FITC
(clone PRB-1) or mouse IgG1-FITC.
water at 0.8 mg/ml from days 6–10 p.i. FLICA reagent was prepared and
added to cells according to manufacturer’s instructions (Thermofisher).
For BrdU staining, cells were permeabilized, treated with 3 μg/mL DNAse
(Invitrogen) and subjected to intracellular staining with either anti-BrdU-FITC
(clone PRB-1) or mouse IgG1-FITC.
4
Assessing Cell Proliferation by BrdU Incorporation
5
Cell Cycle Analysis by BrdU and PI Staining
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Cells were incubated with 3 μg/mL BrdU (Sigma Aldrich) for 1 hour before harvesting via trypsin digestion and fixation with cold 70% ethanol. Cells were permeabilized with 2N HCl and blocked Super Block Buffer (Thermo Scientific) before incubation with anti-BrdU-FITC (Fischer Scientific) overnight. Cells were washed with PBS-T and incubated with 1 mg/mL Propidium Iodide (Sigma) with 0.5 mg/mL RNase (Sigma) before analysis by flow cytometry (BD FACS Canto II).
6
Quantifying Cell Cycle Progression via BrdU Incorporation
7
Quantifying Cell Cycle Progression via BrdU Incorporation
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Ethanol fixed cells were resuspended and washed with 50mM NaCitrate. After mild sonication, cell walls were degraded with 10 units of zymolyase in PBS for 30 min at 30°C, followed by a lysis using 2M HCl and 0.5% Triton Tx-100 for 30 min at room temperature, followed by 30 min incubation at room temperature in 0.1M NaB4O7. Cells were then washed and resuspended in PBS, 1% milk, 0.2% Tween and 1:20 anti-BrdU-FITC (ThermoFisher #11-5071-42) and incubated for 30 min at RT. Cells were again washed and resuspended in PBS, 1% milk, 0.2% Tween, 0.25mg/mL RNase A and 10mg/mL propidium iodide and incubated at 37°C for 30 min. Cells were washed and resonicated before flow cytometry detection using a MACSquant flow cytometer. Data was analyzed using FlowJo software.
8
Cell Cycle Analysis by BrdU and PI Staining
9
Cell Cycle Analysis by Flow Cytometry
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Ethanol fixed cells were resuspended and washed with 50mM NaCitrate. After mild sonication, cell walls were degraded with 10 units of zymolyase in PBS for 30 min at 30°C, followed by a lysis using 2M HCl and 0.5% Triton Tx-100 for 30 min at room temperature, followed by 30 min incubation at room temperature in 0.1M NaB4O7. Cells were then washed and resuspended in PBS, 1% milk, 0.2% Tween and 1:20 anti-BrdU-FITC (ThermoFisher #11-5071-42) and incubated for 30 min at RT. Cells were again washed and resuspended in PBS, 1% milk, 0.2% Tween, 0.25mg/mL RNase A and 10mg/mL propidium iodide and incubated at 37°C for 30 min. Cells were washed and resonicated before flow cytometry detection using a MACSquant flow cytometer. Data was analyzed using FlowJo software.
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