Product Information

ABSOLUTE-S^TM Methodology

Figure 1: Schematic illustration of the ABSOLUTE-S^TM methodology

Incorporation of BrdUrd during DNA replication is followed by cell exposure to UV light. High-energy photons of UV light absorbed by BrdUrd cause DNA photolysis at the sites adjacent to the incorporated BrdUrd. The 3' OH ends in the photolysis-induced DNA breaks are then labeled with Br-dUTP con using TdT as a catalyst. FITC-conjugated anti-BRDU antibody is used to label the Br-dUTP incorporation as in the APO-BRDU^TM (catalog no. 88-6671-88) assay.

The growth of cells in the presence of 5-bromo-2-deoxyuridine (BrdUrd) has become an accepted method for monitoring DNA replication. The BrdUrd incorporation into the cellular DNA can be detected by reporter molecule-labeled antibodies. The use of BrdUrd for assaying DNA replication is replacing methods utilizing radioisotope-labeled thymidine in both research and clinical laboratories. This assay is proving useful for the evaluation of tumor prognosis.

The incorporation of BrdUrd into cellular DNA is most commonly detected using anti-BrdUrd antibodies. This methodology, although proven effective, is limited when it comes to providing a complete analysis of the cells and/or tissues under investigation. This limitation arises as a result of the techniques required to make the incorporated BrdUrd available for measurement. It requires that the cellular DNA be denatured to separate the duplex strands in order for the BrdUrd epitope to become accessible and reactive to the antibody. This denaturation process usually involves either a heat treatment (greater than 90°C) or acid (2-4 N HCl) treatment. The requirement for DNA denaturation results in the loss and/or extraction of many cellular proteins. This fact makes it difficult to combine additional simultaneous assays and probes for cell function and immunophenotyping when utilizing the heat/acid denaturation steps.

The strand break induced photolysis (SBIP) method utilized in the ABSOLUTE-S^TM Kit does not require DNA denaturation and therefore is applicable in studies where preservation of antigens or other cellular features of the cells are desired. The SBIP method is based on the selective photolysis of DNA by ultraviolet (UV) light at sites of incorporation of BrdUrd. After photolysis of the DNA at the sites of BrdUrd incorporation, the UV-induced strand breaks are labeled with a modified deoxynucleotide triphosphate (Bromolated dUTP) by a reaction catalyzed by terminal deoxynucleotidyl transferase (TdT). The sites of BrdUTP incorporation into the DNA stand breaks are then identified with a reporter labeled anti-BrdUTP antibody (Fluorescein~PRB-1 antibody).

ABSOLUTE-S^TM Protocol

Important: The experimental cells to be subject to the ABSOLUTE-S^TM protocol should be in their exponential growth phase. All steps of this procedure should be done in a manner to minimize exposure of the cells to light until after the Strand Break Induced Photolysis of the cells has been accomplished.

1) BrdUrd Incorporation

1. Add 20 µl of the BrdUrd Photolyte stock solution (ASBP13, Pink cap) per 10 ml of cell culture medium.
2. Incubate the cells at 37°C (CO₂ incubator) for 20 to 40 minutes.
3. Following the incubation with the BrdUrd Photolyte, treat cell culture medium with 2% (v/v) dimethyl sulfoxide (DMSO) and immediately add 20 µl of Photolyte Enhancer (ASPE14, Black cap) per 10 ml of cell culture medium and incubate an additional 20 minutes in the dark in the presence of CO₂.
4. Centrifuge the cells for 5 minutes (300xg) and remove the supernatant by aspiration.
5. Resuspend the cells at a concentration of 1-5 x 10⁶ cells/ml in Phosphate Buffered Saline (PBS).

2) Photolysis of DNA

1. Place the cell suspension in a 60 x 15 mm petri dish.
2. Illuminate for 5 minutes using a Fotodyne UV300 analytic DNA transilluminator or equivalent light source. The Fotodyne UV300 contains four 15 W bulbs that emit at 312 nm maximum wavelength. The average UV intensity of the light sources is 4.5 mW/cm².
3. Centrifuge cells for 5 minutes (300xg) remove the supernatant by aspiration.

Note: If multiparameter analysis, i.e., surface receptors and proliferation markers, is to be performed, cell fixation at this point may not be desirable.

3) Fixation of Cells

1. Fix the cells at 1-2 x 10⁶ cells/ml in ice cold 70% (v/v) ethanol.
2. Store cells overnight or for several days until use at minus 20°C.

This completes the Strand Break Induced Photolysis (SBIP) portion of the protocol.

References

1. Xun Li, Frank Traganos, Myron R. Melamed, and Zbigniew Darzynkiewicz, Cytometry, Vol. 20, Number 2, June 1995, pp. 172-180.
2. Pheonix Flow, ABSOLUTE-S Protocol Booklet, http://www.phnxflow.com/pdfs/ABSOLUTSpdf.pdf

ABSOLUTE-S and APO-BRDU are trademarks of Phoenix Flow Systems, San Diego, California.