Human NSCLC cells were seeded at a density of 1×105 cells per 60-mm plate. The following day, bromodeoxyuridine (BrdU; 30 μM; Sigma-Aldrich) diluted in fresh medium was administered to the cells for 30 minutes. The cells were harvested, fixed, and processed for incubation with primary mouse anti-BrdU monoclonal antibody (Roche) and subsequently secondary APC-conjugated goat anti-mouse antibody (Biotium). BrdU-positive cells were counted. Finally, the cells were stained with propidium iodide (Sigma-Aldrich) for flow cytometry analysis. The average of three separate experiments has been documented.
Primary mouse anti brdu monoclonal antibody
Manufactured by Roche
Sourced in United States, Germany
The Primary mouse anti-BrdU monoclonal antibody is a laboratory reagent used for the detection and analysis of cells that have incorporated bromodeoxyuridine (BrdU) during DNA synthesis. It is a specific antibody that binds to BrdU, which can be used as a marker for cell proliferation studies.
Automatically generated - may contain errors
Lab products found in correlation
Bromodeoxyuridine (brdu), by Merck Group (3 mentions)
Propidium iodide, by Merck Group (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Bromodeoxyuridine (brdu), by Zeiss (1 mentions)
Four well chamber slide, by Thermo Fisher Scientific (1 mentions)
Bromodeoxyuridine (brdu), by Roche (1 mentions)
Pro long gold antifade mounting solution with dapi, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
4 protocols using primary mouse anti brdu monoclonal antibody
1
BrdU Incorporation Assay in NSCLC Cells
2
BrdU Incorporation Assay for Proliferation
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Human NSCLC cells were seeded at a density of 1 × 105 cells per 60 mm plate. The following day, BrdU (30 µM; Sigma-Aldrich) diluted in fresh medium was administered to the cells for 30 min. The cells were harvested, fixed and processed for incubation with primary mouse anti-BrdU monoclonal antibody (Roche) and subsequently secondary allophycocyanin (APC)-conjugated goat anti-mouse antibody (Biotium). BrdU-positive cells were counted. Finally, the cells were stained with propidium iodide (Sigma-Aldrich) for flow cytometry analysis. The average of three separate experiments has been documented.
3
Quantifying Cell Proliferation by BrdU Staining
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Cell-proliferative potential was monitored by bromodeoxyuridine (BrdU) staining. Both types of cells were seeded at 5 × 103 cells/cm2 in four-well chamber slides (Thermo Fisher Scientific). On days 6 and 14 post seeding, 10 μM BrdU (Merck KGaA) was added to the cultures, which were then incubated for 3 h at 37 °C prior to fixation. BrdU incorporation was determined with mouse monoclonal anti-BrdU primary antibody (diluted 1:200; Roche Diagnostics) and Alexa 488-conjugated goat polyclonal anti-mouse IgG secondary antibody (diluted 1:1000; Life Technologies, Carlsbad, CA, USA). BrdU-positive cells were counted in 10 random fields under a confocal laser-scanning microscope (LSM 700; Carl Zeiss).
4
Immunofluorescence Staining of BrdU-Labeled Cells
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For immunofluorescence staining, the cells were fixed on coverslips with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min on ice. Following permeabilization with 0.2% Triton X-100 (USB Corp., Cleveland, OH, USA) and blocking solution treatment (5% non-fat milk in PBS with 0.1% Triton X-100) for 30 min at room temperature, the cells were incubated with mouse monoclonal anti-BrdU primary antibody (1:20, #11-170-376-001; Roche Diagnostics GmbH, Mannheim, Germany) for 1.5 h at room temperature. Subsequent to being washed three times with 1% non-fat milk in PBS with 0.1% Triton X-100, the cells were treated with Alexa 488 anti-mouse immunoglobulin G1 secondary antibody (Invitrogen Life Technologies, Carlsbad, CA, USA) for 45 min at room temperature. When necessary, actin was stained with phalloidin (Invitrogen Life Technologies). Finally, the samples were mounted using ProLong® Gold Antifade mounting solution with DAPI (Invitrogen Life Technologies) and left to dry overnight prior to observation. The samples were then examined using a fluorescence microscope (Leica Microsystems Gmbh).
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