Description
Bromodeoxyuridine (BrdU) is an analog of thymidine that can be incorporated into newly synthesized DNA by cells entering and progressing through the DNA synthesis (S) phase of the cell cycle. The amount of incorporated BrdU depends on the amount of time that the cells are exposed to BrdU (pulse time), the rate of cell division, and whether the cells are in early, mid, or late S phase. Investigators can identify cycling cells in an asynchronous cell population and determine cell cycle kinetics by detecting incorporated BrdU. The 3D4 monoclonal antibody reacts with BrdU, but not other nucleotides, in single-stranded DNA. Random cleavage (nicking) of cellular DNA with DNase I permits the binding of the antibody to incorporated BrdU.
Cells are pulse labeled with BrdU, fixed to maintain BrdU loading, and permeabilized for intranuclear staining of the incorporated BrdU. DNase I is used to randomly cleave the DNA, allowing the anti-BrdU monoclonal antibody to bind to the incorporated BrdU. Materials • Adherent cell culture growing in a Falcon™ 96-well Imaging Plate • 37°C incubator • BrdU (Cat. No. 550891) diluted to 10-100 μM in tissue culture medium, prepare enough to use at 50 μl per well (see step 1 of procedure) • BD Cytofix™ fixation buffer (Cat. No. 554655), warmed to 37°C • BD™ Phosflow Perm Buffer III (Cat. No. 558050), at -20°C • Phosphate-buffered saline solution (PBS), at room temperature • BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), at 4°C • 300 μg/ml sterile solution of DNase I (preferred, Sigma-Aldrich Cat. No. D4513) in PBS, prepare enough to use at 50 μl per well • Alexa Fluor® 647 Mouse anti-BrdU monoclonal antibody diluted 1:10 in BD Pharmingen™ Stain Buffer (FBS), prepare enough to use at 50 μl per well. If additional antibodies are to be used, they should be added so that all antibodies are included in the 50 μl per well. • 2 μg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in PBS, prepare enough to use at 100 μl per well • Fluorescence imaging instrument capable of exciting Alexa Fluor® 647 and Hoechst 33342, such as the BD Pathway™ Bioimaging System Procedure 1. Pulse the adherent cell cultures by adding 50 μl of the diluted BrdU to each well and incubating for 1-2 hrs at 37°C. Omit the BrdU from the wells that will be used as negative controls for the BrdU staining. In all subsequent steps, the BrdU-loaded and control cells should be processed in parallel. 2. Remove the medium from the wells, and fix the cells by adding 100 μl of the pre-warmed fixation buffer to each well and incubating for 10 - 20 minutes at room temperature. 3. Remove the fixative from the wells, and wash the wells twice with 100 μl of PBS. 4. Remove the PBS, and permeabilize the cells by adding 100 μl of the ice-cold BD™ Phosflow Perm Buffer III to each well and incubating for 5 - 10 minutes at room temperature. 5. Remove the Perm Buffer III from the wells, and wash the wells twice with 100 μl of PBS. 6. OPTIONAL: Remove the PBS, and block the cells by adding 100 μl of the BD Pharmingen™ Stain Buffer (FBS) to each well and incubating for 15 - 30 minutes at room temperature. 7. Remove the BD Pharmingen™ Stain Buffer (FBS), and denature the cells' DNA by adding 50 μl of the sterile DNase I solution to each well and incubating for 1 hour at 37°C. 8. Remove the DNase I solution from the wells, and wash the wells once with 100 μl of PBS. 9. Remove the PBS, and stain the cells by adding 50 μl of the diluted antibody solution to each well and incubating for 1 hour at room temperature. 10. Remove the antibody solution, and wash the wells twice with 100 μl of PBS. 11. Remove the PBS, and counter-stain the nuclei by adding 100 μl of the Hoechst 33342 solution to each well at least 15 minutes before imaging. 12. View and analyze the cells on an appropriate imaging instrument. Recommended filters for the BD Pathway™ cell analyzers are: Instrument Excitation Emission Dichroic BD Pathway 855 620/60 700/75 660 LP BD Pathway 435 628/40 690/40 FF660
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
1. This reagent has been pre-diluted for use at the recommended Volume per Test when following the Recommended Assay Procedure. A Test is typically ~10,000 cells cultured in a well of a 96-well imaging plate.
2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
4. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
5. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
6. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
