Bromodeoxyuridine Pulse Labelling Assay: A Technique to Measure Cell Proliferation

Abstract

Source: Welschinger R. et al. Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization. J. Vis. Exp. (2015)

The video describes the stepwise pulse labeling assay of mammalian cells with bromodeoxyuridine (BrdU) for measuring cell proliferation. The BrdU uptake permits the temporal tracking of cells that were in the synthesis phase at a specific point in time, without requiring the cells to be synchronized.

Protocol

1. Solutions and Reagents

1. Complete RPMI
   1. 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.
2. BrdU Stock Solution
   1. Prepare 32.5 mM BrdU (10 mg/ml) in Dulbecco's Phosphate Buffered Saline (DPBS).
3. BrdU Complete RPMI
   1. Add 6.2 µl of BrdU stock solution to 10 ml of Complete RPMI.
4. DNase Solution
   1. Prepare 1 mg DNase/ml in DPBS.
5. Staining Buffer
   1. Prepare 3% heat-inactivated FCS and 0.09% sodium azide in DPBS.
6. Refer to Materials List....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NALM6	DSMZ	ACC-128
5-Bromo-2′-deoxyuridine	Sigma	B5002-1G
RPMI 1,640 w/o L-Gln 500 ml	Lonza	12-167F
DPBS	Lonza	17-512F
Fetal Bovine Serum	FisherBiotec	FBS-7100113
CO2 incubator	Binder	C 150
BD Falcon 12 x 75 mm FACS tubes	BD Biosciences	352008
BD LSR FORTESSA flow cytometer	BD Biosciences	FORTESSA
Centrifuge	Spintron	GT-175R