Name: BrdU Immunofluorescence Staining: A Technique to Identify Cells in Different Phases of Cell Cycle
Uploaded: 2023-04-30T13:13:34
Description: Source: Welschinger, R., et al. Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization. J. Vis. Exp. ...

1. Solutions and Reagents
<ol>
	<li>Complete RPMI
	<ol>
		<li>Add 56 ml fetal calf serum (FCS) and 5.5 ml of 200 mM L-glutamine to a 500 ml bottle of RPMI-1640 medium.</li>
	</ol>
	</li>
	<li>DNase Solution
	<ol>
		<li>Prepare 1 mg DNase/ml in DPBS.</li>
	</ol>
	</li>
	<li>Staining Buffer
	<ol>
		<li>Prepare 3% heat-inactivated FCS and 0.09% sodium azide in DPBS.</li>
	</ol>
	</li>
	<li>Refer to Materials List for definitions of Fixation Buffer, Permeabilization Buffer and Wash Buffer.</li>
</ol>
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brdu immunofluorescence staining: a technique to identify cells in different phases of cell cycle

Source: Welschinger, R., et al. <a href="https://www.jove.com/t/52840">Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization.</a> J. Vis. Exp. (2015).
This video describes the protocol for BrdU immunofluorescence staining. The technique allows the identification of cells in different phases of cell cycle. Addition of DNA dyes and antibody labeling allows the detailed analysis of the fate of the S phase cells at later times.

BrdU Immunofluorescence Staining: A Technique to Identify Cells in Different Phases of Cell Cycle

Source: Welschinger, R., et al. Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization. J. Vis. Exp. ...

- Bromodeoxyuridine or BrdU is a synthetic thymidine nucleoside analog. During the synthesis phase of the cell cycle, BrdU substitutes thymidine and integrates into the newly synthesized DNA. The incorporated BrdU allows the tracking of cell-cycle progression. To detect the incorporated BrdU, first, fix and permeabilize the cells to render them permeable.
Next, treat the cells with DNAse enzyme to cleave the DNA strands and expose the incorporated BrdU. Subsequently, add a suitable fluorescent labeled anti-BrdU antibody. The antibody enters the nucleus and readily binds to the exposed BrdU present in the DNA.
Additionally, treat the cells with a suitable fluorescent DNA-binding dye. The dye binds to the DNA, allowing the quantification of total DNA content. Use flow cytometry to detect the fluorescence from both labeled BrdU and DNA.
Fluorescence intensities from the labeled DNA differentiate cells in different cell-cycle phases based on the variation in total DNA content. The fluorescent signal from BrdU identifies the current cell cycle position of BrdU-incorporated cells.
In this example, we will perform the BrdU immunofluorescent staining of NALM-6-- acute lymphoblastic leukemia cells.
- Prior to antibody staining, treat the cells with DNAse. Resuspend the cells in 100 microliters of DNAse solution and incubate for one hour at 37 degrees Celsius. Add one milliliter of wash buffer, centrifuge the cells, and discard the supernatant. Antibody staining for intracellular markers other than BrdU can be performed simultaneously with the BrdU staining.
- It&#39;s important to prepare compensation controls consisting of unstained cells and cells labeled with each single fluorochrome. Ideally, use the same antibodies for compensation controls as those used in the experimental tubes.
- Resuspended the cells in 50 microliters of wash buffer with one microliter per 10 to the 6 cells of BrdU antibody. If using antibodies to other specific intracellular antigens, they should be added at this point. Incubate the cells for 20 minutes at room temperature.
After 20 minutes, wash the cells with one milliliter of wash buffer as shown earlier. Finally, stain the DNA for cell-cycle analysis. Loosen the pellet by flicking the tube and add 20 microliters of the 7-AAD solution. It is critical to use a constant amount of 7-AAD per cell. Resuspend the cells in one milliliter of staining buffer. Subsequently, perform flow cytometry analysis.

brdu-immunofluorescence-staining-technique-to-identify-cells

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Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

BrdU Immunofluorescence Staining: A Technique to Identify Cells in Different Phases of Cell Cycle

Transcript