Cells were seeded in 6-well plates at 3x105 cells/well and treated with 5 μM olaparib, 5 nM prexasertib, both or 0.01% DMSO. Cells were treated with indicated drugs for 24 hours and 48 hours for cell cycle analysis. Briefly, cells were fixed in 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton-X for 5 minutes, and blocked with 10% goat serum in PBS for 30 minutes. Cells were incubated in primary and secondary antibodies for 1 hour each at 4°C. Cellular DNA was stained using 7-AAD. For primary antibodies, anti-mouse phospho-Histone H3 (Ser10) (Cell Signaling, #9701) at 1:50 ratio in 10% goat serum/PBS was used. Alexa Fluor 488nm goat anti-rabbit secondary antibodies (Invitrogen) and Alexa Fluor 647nm goat anti-mouse secondary antibodies (Invitrogen) were used at 1:500 dilutions. Cell cycle analysis was performed using the BD Pharmingen APC BrdU flow kit according to the manufacturer’s protocol (BD Biosciences; San Jose, CA, USA). Stained cells were collected with a FACScalibur (BD Biosciences) and analyzed using the FlowJo v. X.0.8 software (Treestar; Ashland, OR, USA).
Pharmingen apc brdu flow kit
Manufactured by BD
Sourced in United States
The BD Pharmingen APC-BrdU Flow Kits are laboratory equipment designed for the detection and analysis of DNA synthesis and cell proliferation using flow cytometry. The kits provide the necessary reagents and protocols to label and detect bromodeoxyuridine (BrdU) incorporation into cellular DNA.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6 flow cytometer, by BD (3 mentions)
Bromodeoxyuridine (brdu), by BD (2 mentions)
Facscalibur, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Annexin 5 fitc apoptosis kit, by BioLegend (1 mentions)
Bromodeoxyuridine (brdu), by BioLegend (1 mentions)
Phase flow brdu cell proliferation kit, by BioLegend (1 mentions)
Facscanto 2, by BD (1 mentions)
7 protocols using pharmingen apc brdu flow kit
1
Cell Cycle Analysis of Olaparib and Prexasertib
2
Cell Cycle Analysis of Mouse Bone Marrow Cells
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Cell cycle analysis was performed using the BD Pharmingen APC BrdU Flow Kit (BD Bioscience) according to the manufacturer’s protocol.
Briefly, 1x106 transduced mouse BM cells were cultured for 48h prior to labeling with 10 μM BrdU for 1 hour. Cells were harvested, washed once with Staining Buffer (1x PBS supplemented with 3% h.i. FCS and 0.09% sodium azide) and incubated with Cytofix/Cytoperm buffer for 15 min on ice. After washing with PermWash buffer, cells were permeabilized with Cytoperm Plus buffer for 10 min on ice, washed with PermWash buffer, incubated again in Cytofix/Cytoperm buffer for 5 min on ice followed by another PermWash buffer washing step. BrdU epitopes were then exposed by treating the cells with 0.4 mg/ml DNase I for 1 hour at 37°C. After removal of the DNase by washing the cells with PermWash buffer, they were incubated with 1 μl APC-conjugated BrdU antibody in 50 μl PermWash buffer for 20 min at RT in the dark. Subsequently, cells were subjected to a final PermWash washing step, resuspended in 20 μl 7-AAD and incubated for 5 min at RT in the dark. After addition of 1 ml Staining Buffer, cells were analysed using a BD LSRFortessa flow cytometer.
Briefly, 1x106 transduced mouse BM cells were cultured for 48h prior to labeling with 10 μM BrdU for 1 hour. Cells were harvested, washed once with Staining Buffer (1x PBS supplemented with 3% h.i. FCS and 0.09% sodium azide) and incubated with Cytofix/Cytoperm buffer for 15 min on ice. After washing with PermWash buffer, cells were permeabilized with Cytoperm Plus buffer for 10 min on ice, washed with PermWash buffer, incubated again in Cytofix/Cytoperm buffer for 5 min on ice followed by another PermWash buffer washing step. BrdU epitopes were then exposed by treating the cells with 0.4 mg/ml DNase I for 1 hour at 37°C. After removal of the DNase by washing the cells with PermWash buffer, they were incubated with 1 μl APC-conjugated BrdU antibody in 50 μl PermWash buffer for 20 min at RT in the dark. Subsequently, cells were subjected to a final PermWash washing step, resuspended in 20 μl 7-AAD and incubated for 5 min at RT in the dark. After addition of 1 ml Staining Buffer, cells were analysed using a BD LSRFortessa flow cytometer.
3
Cell Cycle and Apoptosis Analysis
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Cells were incubated with 50 mM BrdU (BD Bioscience, Franklin Lakes, NJ, USA) for 2 h, and cell cycle analysis was performed using BD Pharmingen APC-BrdU Flow Kits according to the manufacturer's protocol (BD Bioscience, Franklin Lakes, NJ, USA). The samples were analyzed by flow cytometry on a BD Accuri C6 Flow Cytometer (BD Bioscience). Cell apoptosis was detected using the Annexin V-FITC Apoptosis Kit (Biolegend, San Diego, CA) according to the manufacturer's protocol. The cells were cultured for 48 h with normal medium and then treated with or without drugs for 24 h. Cells were collected and re-suspended in 1× binding buffer at a concentration of 1 × 106 cells per mL. Then, the cells (1 × 105) were incubated with 5 μL of Annexin V-FITC and 5 μL of PI for 15 min at 24 °C in the dark, followed by the addition of another 400 μL of 1× binding buffer. Samples were analyzed by flow cytometry. The data were analyzed using FlowJo software (San Francisco, CA, USA).
4
BrdU Labeling and Cell Cycle Analysis
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BrdU (1 mM) in GK15 was diluted in GK15+BSELY medium to obtain a final concentration of 20 μM. Then half the medium of hPGCLC cultures in V-bottom plates was changed (final BrdU concentration of 10 μM). BrdU-treated aggregates were incubated at 37°C under 5% CO2 in air for 1 h. hPGCLCs were dissociated into single cells, and cell cycle analysis was performed by staining cells with BrdU-APC and 7-AAD with BD Pharmingen APC BrdU Flow Kits (552598, BD Biosciences), according to the manufacturer’s instructions. Stained cells were analyzed in FACSFortessa.
5
Cell Cycle Analysis via BrdU Incorporation
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Cells were incubated with 50 μM BrdU (BD Bioscience) for 1 h and cell cycle analysis was performed using BD Pharmingen APC-BrdU Flow Kits according to the manufacturer's protocol (BD Bioscience, NJ, USA). The samples were analysed by flow cytometry on a BD Accuri C6 Flow Cytometer (BD Bioscience). The following cell cycle phases were determined as a percentage of the total population: sub-G0 (apoptotic cells), G0/G1 (2n, BrdU-negative), S (2n to 4n, BrdU-positive) and G2/M phase (4n, BrdU-negative). In some experiments, BrdU (1 mg, BD Bioscience, NJ, USA) was i.p. injected into mice 18 h before mice were killed. The primary tumour cells were isolated from tumour or ascites and used for cell cycle analysis.
6
Quantification of Neural Progenitor Proliferation
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NPCs were incubated with 10 µM BrdU (BD Biosciences #51–2420KC) in a complete embryonic NeuroCult™ proliferation medium. Following 45 min, the NPCs were washed with phosphate-buffered saline (PBS; Sartorius #02-023-1A), detached with ACCUTASE, and stained against Nestin (1 µg per 1 × 106 cells, SANTA CRUZ #sc-33677PE) and BrdU (BD Biosciences #51-51-23619L) using BD Pharmingen™ APC BrdU Flow Kits (BD Biosciences #552598) according to the manufacturer’s protocol. The percentage of BrdU-positive (BrdU+) and Nestin-positive (Nestin+) NPCs were measured separately by fluorescence-activated cell sorter (FACS; BD FACSCantoII, BD Biosciences) and analyzed with FlowJO_v10.8.1 software.
For cell cycle analysis, a separate experimental cohort of cells was washed with PBS and detached with ACCUTASE after a 45 min BrdU pulse (BioLegend #77528). Then, cells were stained with anti-BrdU antibody and 7-AAD utilizing the Phase-Flow™ BrdU Cell proliferation Kit (BioLegend #370704) according to the manufacturer’s protocol. We monitored the fluorescence labeling by FACS and analyzed it with FlowJO_v10.8.1 software to define the percentage of cells in each cell cycle phase: G0/G1, S-phase, and G2/M (Fig.2A ).
For cell cycle analysis, a separate experimental cohort of cells was washed with PBS and detached with ACCUTASE after a 45 min BrdU pulse (BioLegend #77528). Then, cells were stained with anti-BrdU antibody and 7-AAD utilizing the Phase-Flow™ BrdU Cell proliferation Kit (BioLegend #370704) according to the manufacturer’s protocol. We monitored the fluorescence labeling by FACS and analyzed it with FlowJO_v10.8.1 software to define the percentage of cells in each cell cycle phase: G0/G1, S-phase, and G2/M (Fig.
7
BrdU-Based Cell Cycle Analysis
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Cells were incubated with 50 mM BrdU (BD Bioscience, NJ, USA) for 1 h and cell cycle analysis was performed using BD Pharmingen APC-BrdU Flow Kits according to the manufacturer's protocol (BD Bioscience, NJ, USA). The samples were analysed by ow cytometry on a BD Accuri C6 Flow Cytometer (BD Bioscience). In some experiments, BrdU (1 mg, BD Bioscience, NJ, USA) was i.p. injected into mice 18 h before mice were killed. The primary tumor cells were isolated from tumor or ascites and used for cell cycle analysis.
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