ࡱ> /1. bjbj$$ .F|F| """" ."B(jjjjEEErtttttt$dEEEEEjj___Ejjr_Er__&jjW"^0,y&&8EE_EEEEE+4EEEEEEEEEEEEEEEE : This method is one of the original BRDU protocols. Newer, but more expensive, BRDU kits are available from Ebioscience ( HYPERLINK "http://media.ebioscience.com/data/pdf/best-protocols/brdu-staining-for-flow-cytometry.pdf" http://media.ebioscience.com/data/pdf/best-protocols/brdu-staining-for-flow-cytometry.pdf) and Life Technologies. ( HYPERLINK "http://www.lifetechnologies.com/us/en/home/references/protocols/cell-and-tissue-analysis/flow-cytometry-protocol/cell-proliferation/standard-click-it-edu-flow-cytometry-cell-proliferation-assay.html" http://www.lifetechnologies.com/us/en/home/references/protocols/cell-and-tissue-analysis/flow-cytometry-protocol/cell-proliferation/standard-click-it-edu-flow-cytometry-cell-proliferation-assay.html) BRDU with HCL Materials needed 5-bromo-2-deoxyuridine from Sigma Cat # B-5002 Anti-bromodeoxyuridine from Boehringer Mannheim Cat # 11170 376 50mg** Anti-Mouse IgG Sigma Cat # F-2883 100% ETOH Rnase Propidium Iodide Tween Method for one million cells: Make BrdU stock of 9mg/ml final concentration. Store at 4 degrees in the dark. Protect from light during storage as well as during the experiment. Can be reused. Add BrdU to cells at a final concentration of .018 mg/ml and incubate for 30 minutes in the dark in a 37 degree incubator. Rinse 3X with PBS (pH 7.4) Harvest cells as usual. Resuspend cell pellet with 1 ml of PBS. Be sure that cells are in a single cell suspension. Add 2 mls of cold (freezer-temp) 100% ETOH dripwise while vortexing the cells and place at -20 degrees for at least one hour. Centrifuge cells at 1500 rpm for 5 minutes to remove the ETOH. Resuspend the pellet in 1 ml of 2N HCL. Incubate for 30 minutes at room temperature. Wash in 1 ml of PBS/Tween (0.5% Tween v/v) until pH reaches 7.2. Usually takes 2-3 washes. Use pH paper to test. Pellet resuspension in primary BrdU antibody at a final concentration of 10ug/ml in PBS/Tween. Incubate 30 min at room temperature. Wash the cells 3X in PBS/Tween as above. Resuspend the pellet in the secondary antibody diluted in PBS/Tween to a final concentration of 10ug/ml. Incubate 30 min at room temperature. Wash the cells 3X in PBS/Tween as above. Resuspend the pellet into 850 ul of PBS. 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