BD Pharmingen™ APO-BRDU™ Kit

BrdU

One of the later steps in apoptosis is DNA fragmentation, a process which results from the activation of endonucleases during the apoptotic program. These nucleases degrade the higher order chromatin structure into fragments of ~300 kb and subsequently into smaller DNA pieces of about 50 bp length. A method which is often used to detect fragmented DNA utilizes a reaction catalyzed by exogenous TdT, often referred to as "end-labeling" or "TUNEL" (terminal deoxynucleotidyltransferase dUTP nick end labeling). In the APO-BRDU™ assay, TdT catalyzes a template-independent addition of bromolated deoxyuridine triphosphates (Br-dUTP) to the 3'-hydroxyl (OH) termini of double- and single-stranded DNA. After incorporation, these sites are identified by flow cytometric means by staining the cells with a FITC-labeled anti-BrdU mAb. The APO-DIRECT™ assay (Cat. No. 51-6536KK) is a single-step method for labeling DNA breaks with FITC-dUTP, followed by flow cytometric analysis. The APO-BRDU™ Kit is a two color staining method for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry. Sufficient reagents are provided to process 60 cell suspensions. The kit includes 5 ml of positive and 5 ml of negative control cell suspensions, approximately 1 x 10^6 cells per ml in 70% (v/v) ethanol. The control cells are derived from a human lymphoma cell line and have been fixed as described in the Fixation Protocol. The APO-BRDU™ Kit consists of two parts (part A and B).   Part A (Comp. No. 51-6576AK) consists of:   One 0.30 ml bottle of FITC-Labeled Anti-BrdU mAb [Isotype: mouse; 1 µg/test (contains 0.05% sodium azide)] (Comp. No. 51-6584AZ] One 30 ml bottle of PI/RNase Staining Buffer (5 µg/ml, 200 µg/ml RNase) [Comp. No. 51-6585AZ] One 0.6 ml vial of Reaction Buffer (containing cacodylic acid (dimethylarsenic)) [Comp. No. 51-6580AZ ] One 126 ml bottle of Rinsing Buffer (containing 0.05% sodium azide) [Comp. No. 51-6583AZ] One 120 ml botle of Wash Buffer (containing 0.05% sodium azide) [Comp. No. 51-6579AZ].   Part B (Comp. No. 51-6576BK) consists of:   One 0.48 ml bottle of Br-dUTP (54.7 µg/ml)  [Comp. No. 51-6582EZ] One 5 ml bottle of Negative Control Cells [contains 70% (v/v) ethanol] [Comp. No. 51-6578LZ] One 5 ml bottle of Positive Control Cells [contains 70% (v/v) ethanol] [Comp. No. 51-6577LZ} One 0.045 ml bottle of TdT Enzyme [200 µg/ml (S.A.= 100,000 U/mg) in 50% (v/v) glycerol solution; will not freeze at -20°C] [Comp. No. 51-6581EZ]

(RUO)

51-6576AK/(2 to 8C)+51-6576BK/(max.-20C)

Flow cytometry, Intracellular staining (flow cytometry) (Tested During Development)

BrdU