Cells were pulsed with 10 μM bromodeoxyuridine (BrdU) for one hour. Cells were then fixed and stained for BrdU and DNA content with an anti-BrdU FITC-conjugated antibody and with a 7-aminoactinomycin D (7-AAD) dye, respectively, according to the instructions of the BD Pharmingen FITC BrdU Flow Kit (BD Biosciences FITC BrdU Flow Kit 559619, 557891). All data were collected using the BD FACSCalibur software, and results were analyzed by flow cytometry (Cell Lab Quanta™ SC Flow Cytometer, Beckman Coulter).
Fitc brdu flow kit
Manufactured by BD
Sourced in United States
The FITC BrdU Flow Kit is a laboratory tool designed to detect and quantify cell proliferation through the measurement of bromodeoxyuridine (BrdU) incorporation into cellular DNA. The kit provides the necessary reagents to label, detect, and analyze BrdU-positive cells using flow cytometry.
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Lab products found in correlation
Bromodeoxyuridine (brdu), by BD (25 mentions)
Cytofix cytoperm buffer, by BD (18 mentions)
Bromodeoxyuridine (brdu), by Merck Group (18 mentions)
Facscalibur flow cytometer, by BD (15 mentions)
Facscanto 2, by BD (11 mentions)
Facscalibur, by BD (11 mentions)
Lsrfortessa, by BD (9 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (7 mentions)
Perm wash buffer, by BD (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Facsdiva software, by BD (6 mentions)
Fitc annexin 5 apoptosis detection kit 1, by BD (5 mentions)
Cellquest software, by BD (5 mentions)
Cytoperm permeabilization buffer plus, by BD (5 mentions)
Lsrii flow cytometer, by BD (5 mentions)
Lsr 2 flow cytometer, by BD (5 mentions)
Facsverse, by BD (4 mentions)
Facscanto, by BD (4 mentions)
Facsaria 2, by BD (4 mentions)
7 aad, by BD (4 mentions)
297 protocols using fitc brdu flow kit
1
BrdU Cell Cycle Analysis by Flow Cytometry
2
Measuring BrdU Incorporation in Microglia
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The incorporation of 5-bromo-2′-deoxyuridine (BrdU) was analyzed in mixed glial cell cultures using flow cytometry and labeling with a conjugate anti-BrdU antibody (FITC BrdU flow kit, BD Pharmingen, CA, USA). Mixed glial cell cultures were incubated with 0.25–5 μM teriflunomide and co-treated with GM-CSF (5 ng/ml; Peprotech, Hamburg, Germany) from day 5 after preparation. The next day, 10 μM BrdU (FITC BrdU flow kit, BD Pharmingen) was added for 16 h.
After microglia isolation, the Fc receptors were blocked with mouse anti-rat CD32 for 30 min on ice (clone: D34-485; BD Pharmingen). Surface staining of microglia was done with allophycocyanin (APC)-conjugated rat anti-CD11b/c antibody for 30 min at 4 °C. Cells were then fixed and permeabilized with fixation/permeabilization buffer (Foxp3 Staining Buffer Set; eBioscience) according to the manufacturer’s instructions. The samples were treated with DNase to expose incorporated BrdU (diluted to 300 μg/ml; BrdU flow kit, BD Pharmingen) for 1 h at 37 °C and finally stained with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody (30 min at RT, 1:100; BrdU flow kit, BD Pharmingen). The analysis was carried out using a FACSCalibur with CellQuest software (BD). Measurements were performed in duplicates per condition and in four independent experiments.
After microglia isolation, the Fc receptors were blocked with mouse anti-rat CD32 for 30 min on ice (clone: D34-485; BD Pharmingen). Surface staining of microglia was done with allophycocyanin (APC)-conjugated rat anti-CD11b/c antibody for 30 min at 4 °C. Cells were then fixed and permeabilized with fixation/permeabilization buffer (Foxp3 Staining Buffer Set; eBioscience) according to the manufacturer’s instructions. The samples were treated with DNase to expose incorporated BrdU (diluted to 300 μg/ml; BrdU flow kit, BD Pharmingen) for 1 h at 37 °C and finally stained with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody (30 min at RT, 1:100; BrdU flow kit, BD Pharmingen). The analysis was carried out using a FACSCalibur with CellQuest software (BD). Measurements were performed in duplicates per condition and in four independent experiments.
3
Assessing Colitis-Induced Proliferation
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Mice were subjected to DSS protocol to induce colitis, and then injected with BrdU i.p. (1 mg per 20 g body weight) 3 h before killing them. Splenic cells were stained for CD11b+, Gr-1+ and intracellular BrdU staining was performed by using FITC BrdU flow kit (BD Pharmigen, San Diego) according to manufacturer's instructions.
4
Apoptosis and Cell Cycle Analysis
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Cell apoptosis was detected by Annexin V‐FITC Apoptosis Detection Kit (KeyGEN BioTECH, China) following the manufacturer's instruction and then analysed with CytoFLEX (Beckman Coulter, Brea , CA, USA). Cell cycle assay was carried out using PI/RNase Staining Kit (Dojindo) and FITC BrdU Flow Kit (BD Pharmigen, San Diego, CA, USA) in accordance with the manufacturer's instruction. Flow cytometry analysis was performed using CytoFLEX (Beckman Coulter).
5
Cell Cycle Analysis of Ibrutinib Treated Cells
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MD-901 and SUDHL4 cells transduced with pTRIPZ-scramble or pTRIPZ-miR-28 were induced with doxycycline (0.5 μg/ml) for 24 h. Cells were then plated at 200,000 cells/ml and treated with the indicated ibrutinib concentrations in medium containing doxycycline. After 20 h, the cell cycle was analyzed using DAPI staining and the FITC BrdU Flow kit (BD Pharmigen, Cat#559619). The sub G0-G1 population was determined in DAPI-negative gated cells.
6
Cell Cycle and Apoptosis Analysis in MM
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An FITC BrdU Flow kit (BD Pharmigen) was used for cell cycle analysis in MM cells, and an FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen) was used for the detection of apoptotic MM cells, in accordance with the manufacturer’s instructions. Flow cytometry was performed using a BD FACS Canto II (BD Biosciences), and the obtained data were analyzed using FlowJo software (Tree Star).
7
Measuring Cell Proliferation with ReACp53
8
Immunocytochemistry Staining and BrdU Incorporation
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Immunocytochemistry was performed in 96 well plates. Cells were fixed and stained as previously described [62 (link)] with antibodies to p21 (rabbit polyclonal, Cell Signaling # 2947; 1:400 dilution) or Ki67 (mouse monoclonal, Santa Cruz # SC23900; 1:100 dilution) and with appropriate secondary AF647 antibodies (Invitrogen #A-21238 & #A-31571; 1:500 dilution). Nuclei were co-stained with Hoechst 33342. BrdU incorporation experiments were performed as per manufacturers instructions (BD FITC BrdU Flow Kit #559619) and acquired on a LSRII (BD). Analysis was performed using FlowJo software.
9
Isolation and Analysis of Adipose-Derived Stromal Vascular Cells
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Fat depots were collected into 5mL of 1.0 mg/ml collagenase in ADB (w/o glucose and adenosine), minced with scissors and incubated in 37° water bath with shaking for 75 min, shook vigorously by hand and passed through 70um filter into 5mL of KRP buffer. Samples were spun at 300 g for 5min and processed for BRDU labeling using BD FITC BrdU Flow Kit (BD 559619). Briefly, The SVC pellet was resuspended in 200ul staining buffer and incubated with Anti-CD31 PE-Cy7 (BD Biosciences #561410), anti-CD45 PE-CF594 (BD Biosciences #562420) and Anti-CD-140a/PDGFRA, FITC (eBioscience, #11-1401-82) at RT (in dark) for 45min. Then, the samples were washed with 1ml of BD Perm/Wash buffer, spun at 300 g and the pellet resuspended and incubated for 30 min in 100ul in BD Cytofix/Cytoperm buffer. After one additional wash with BD Perm/Wash buffer, cells were resuspended and incubated in 100ul diluted DNase (30ul DNase + 70ul dPBS) at 37°C for 1hr. Then, the samples received BD BRDU antibody (BD 559619) and were incubated at 4°C overnight. Next, the cells were washed with 1ml BD Perm/Wash buffer, spun, resuspended with 250ul of staining buffer, passed through 70um filter and analyzed by flow cytometry, together with unstained and single stained controls.
10
BrdU Incorporation Assay for Cell Cycle
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We used the BD FITC BrdU Flow Kit for the determination of percentage of cells in S phase following the manufacturer's protocol. In brief, at the end of p-PD treatment, cells were incubated with BrdU for 15 minutes and then they were fixed by BD Cytofix/Cytoperm buffer. After that, cells were treated with BD Cytoperm™ Permeabilization Buffer Plus Cells were again incubated with BD Cytofix/Cytoperm buffer followed by a DNase treatment to expose BrdU epitopes. Immunofluorescent staining was done using fluorochrome conjugated anti-BrdU antibody. Finally, cells were stained with 7-AAD followed by analysis in BD FACSVerse using BD FACSuite software. At least 20,000 events were recorded and analysed.
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