BD, 560210, BD Pharmingen™ Alexa Fluor® 555 Mouse anti-BrdU

Alternative Name: 5-bromo-2'-deoxyuridine, 5-Bromouracil deoxyriboside, BUdR
Isotype: Mouse IgG1, κ
Application: Bioimaging (Routinely Tested)
Vol. Per Test: 5 µl
RRID: AB_1645614
Storage Buffer: Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
Regulatory Status: RUO
Preparation And Storage: Preparation And Storage 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 the dye under optimum conditions and unreacted dye was removed.
Recommended Assay Procedures: Recommended Assay Procedures 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 BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) • 37 ° C incubator • BrdU (Cat. No. 550891) diluted to 10-100 μ M in tissue culture medium, prepare enough to use at 50 μ l 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 μ g/ml sterile solution of DNase I (preferred, Sigma-Aldrich Cat. No. D4513) in PBS, prepare enough to use at 50 μ l per well • Alexa Fluor® 555 Mouse anti-BrdU monoclonal antibody diluted 1:10 in BD Pharmingen™ Stain Buffer (FBS), prepare enough to use at 50 μ l per well.  If additional antibodies are to be used, they should be added so that all antibodies are included in the 50 μ l per well. • 2 μ g/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in PBS, prepare enough to use at 100 μ l per well • Fluorescence imaging instrument capable of exciting Alexa Fluor® 555 and Hoechst 33342, such as the BD Pathway™ Bioimaging System Procedure 1. Pulse the adherent cell cultures by adding 50 μ l 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 μ l 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 μ l of PBS. 4. Remove the PBS, and permeabilize the cells by adding 100 μ l 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 μ l of PBS. 6. OPTIONAL: Remove the PBS, and block the cells by adding 100 μ l 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 μ l 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 μ l of PBS. 9. Remove the PBS, and stain the cells by adding 50 μ l 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 μ l of PBS. 11. Remove the PBS, and counter-stain the nuclei by adding 100 μ l 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 855 548/20 570LP Fura/FITC BD Pathway 435 543/22 593/40 FF562