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REACTIVITY SENSITIVITY MW (kDa) Isotype
All Mouse IgG1
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Flow Cytometry

Flow cytometric analysis of Jurkat cells incorporated with BrdU (30 min), using BrdU (Bu20a) Mouse mAb (PE conjugate) versus DRAQ5® (DNA content).

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Flow Cytometry

A. Solutions and Reagents

  1. 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.
  2. BrdU (5-Bromo-2′-deoxyuridine) EMD biosciences (Cat. #203806)
  3. Ethanol, anhydrous denatured, histological grade, 100% and 95%
  4. 1.5 M Hydrochloric acid
  5. Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100 mL 1X PBS. Store at 4°C.
  6. Recommended Anti-Mouse secondary antibodies::

B. BrdU Incorporation and Specimen Preparation

  1. Add BrdU to fresh, warm media for a final concentration of 0.03 mg/mL. Set aside.
  2. Collect ~50 million cells in tube by centrifugation and aspirate supernatant.
  3. Add 2 ml of BrdU-containing media to cell pellet, vortex, and incubate at 37°C for 30 minutes.
  4. Pellet cells by centrifugation and aspirate media.
  5. Add 2 ml of cold, 70% ethanol to cell pellet and mix.
  6. Allow cells to fix for 5 minutes at room temperature.
  7. Add 2–3 ml PBS and rinse by centrifugation three times.
  8. Add 1.5 M HCL and incubate for 30 minutes at room temperature.
  9. Add 2–3 ml PBS and rinse by centrifugation two times.
  10. Proceed with Immunostaining section C.

C. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemacytometer or alternative method.

  1. Aliquot 0.5–1 x 106 cells into each assay tube (by volume).
  2. Add 2–3 ml Incubation Buffer to each tube and rinse by centrifugation. Repeat.
  3. Resuspend cells in 100 µl Incubation Buffer per assay tube.
  4. Block in Incubation Buffer for 10 minutes at room temperature.
  5. Add the unconjugated primary antibody at the appropriate dilution to the assay tubes (see antibody data sheet for the appropriate dilution).
  6. Incubate for 1 hour at room temperature.
  7. Rinse as before in Incubation Buffer by centrifugation.
  8. Resuspend cells in fluorochrome-conjugated secondary antibody diluted in Incubation Buffer at the recommended dilution.
  9. Incubate for 30 minutes at room temperature.
  10. Rinse as before in Incubation Buffer by centrifugation.
  11. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to step D1.

D. Optional DNA Stain

  1. Resuspend cells in 0.5 ml of DNA dye (eg. DRAQ5® #4084).
  2. Incubate for at least 5 mins at room temperature.
  3. Analyze cells in DNA stain on flow cytometer.

posted December 2009

Protocol Id: 30

Product Usage Information

Application Dilutions
Flow Cytometry 1:50

Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

BrdU (Bu20a) Mouse mAb (PE Conjugate) detects BrdU when incorporated into single stranded DNA. DNA must be denatured for the epitope to be exposed and recognized by the antibody.


Species Reactivity: All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with BrdU conjugated to BSA.

Product Description

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated BrdU (Bu20a) Mouse mAb #5292.


Halogenated nucleotides such as the pyrimidine analog bromodeoxyuridine (BrdU) are useful for labeling nascent DNA in living cells and tissues. BrdU becomes incorporated into replicating DNA in place of thymidine and subsequent immunodetection of BrdU using specific monoclonal antibodies allows labeling of cells in S phase of the cell cycle. After pulse-labeling cells or tissues with bromodeoxyuridine, BrdU (Bu20a) Mouse mAb can be used to detect BrdU incorporated into single stranded DNA. Please see our detailed protocol for information regarding the labeling procedure and denaturation of double stranded DNA for various immunodetection applications (1-4).


1.  Darzynkiewicz, Z. and Juan, G. (2001) Curr Protoc Cytom Chapter 7, Unit 7.7.

2.  Leif, R.C. et al. (2004) Cytometry A 58, 45-52.

3.  Staszkiewicz, J. et al. (2009) Biochem Biophys Res Commun 378, 539-44.

4.  Rothaeusler, K. and Baumgarth, N. (2007) Curr Protoc Cytom Chapter 7, Unit7.31.



For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.

50230
BrdU (Bu20a) Mouse mAb (PE Conjugate)