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

Abstract

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

Protocol

1. Solutions and Reagents Complete RPMI 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. DNase Solution Prepare 1 mg DNase/ml in DPBS. Staining Buffer Prepare 3% heat-inactivated FCS and 0.09% sodium azide in DPBS. Refer to Materials List for definitions of Fixation Buffer, Permeabilization Buffer and Wash Buffer. …

Materials

APC BrdU Flow Kit	BD Biosciences	552598	Contains BrdU antibody, 7-AAD and BD Cytofix/Cytoperm Buffer (referred to as Fixation Buffer)
BD Perm/Wash Buffer	BD Biosciences	554723	Referred to as Wash buffer
BD Pharmingen Stain Buffer	BD Biosciences	554656
Pipetman	Gilson	P2, P20, P100, P1000
DNase	Sigma	D-4513
BD Cytoperm Permeabilization Buffer Plus	BD Biosciences	561651	Referred to as Permeabilization buffer
Centrifuge	Spintron	GT-175R
AF488 anti-Histone H3 Phospho (Ser10) Antibody	Cell Signalling	9708S
Phospho-Chk2 (Thr68) (C13C1) Rabbit mAb	Cell Signalling	2197S
Phospho-Chk1 (Ser345) (133D3) Rabbit mAb	Cell Signalling	2348S
BD Falcon 12 x 75 mm FACS tubes	BD Biosciences	352008