The experimental cells to be subject to the ABSOLUTE-S™ 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 and Permeabilization



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 37C (CO₂ incubator) for 20 to 40 minutes.

3. Following the incubation with the BrdUrd Photolyte add dimethyl sulfoxide(DMSO) to the cell culture medium to achieve a final concentration of 2% (v/v) DMSO and then immediately add 20 µl of Photolyte Enhancer (ASPE14, Black cap) per 10 ml of cell culture medium. Incubate an additional 20 minutes in the dark at the incubator.

4. Centrifuge the cells for 5 minutes (300 x g) and remove the supernatant by aspiration.

5. Resuspend the cells at a concentration of 1 to 5 x 10⁶ cells/ml in Wash Buffer (blue cap).

6. Repeat Step D.

7. Resuspend the cell pellet in the tube in the residual Wash Buffer (blue cap) left after aspiration by gently vortexing the tube.

8. Adjust the cell concentration to 1-2 x 10⁶ cells/ml in 70% (v/v) ice cold ethanol.

9. Store the cells protected from light in the freezer over night or until ready to use (at least 18 hours).

 

2. Photolysis of DNA of the Experimental cells and control cells
The control cells supplied with the kit should be photolyzed at the same time as the experimental cell sample.


1. Swirl the bottles containing the positive control cells and the negative control cells to resuspend the cells. Transfer 1 ml of the cell suspension to 12 x 75 mm polystyrene test tubes.

2. Transfer 1 ml of the BrdUrd incorporated experimental cells obtained in part 1 to appropriately labeled 12x75 mm polystyrene test tubes. (If large numbers of cells need to be irradiated, this step may be carried out in a 60x15 mm polystyrene petri dish.

3. Add 2 ml of Wash Buffer (blue cap) and centrifuge (300 x g) for 5 minutes. Remove the supernatant by aspiration being careful to not disturb the cell pellet.

4. Place the test tube containing the experimental cells and the tubes containing the positive and negative control cells on the irradiating surface of the light box. Illuminate the cells for 5 minutes using a Fotodyne UV300 analytic DNA transilluminator or equivalent light source. The Fotodyne UV300 contains four 15W bulbs that emit at 312nm maximum wavelength. The average UV intensity of the light sources is 4.5 mW/cm².

5. After illumination, add 1 ml of Wash Buffer to each tube and centrifuge cells for 5 minutes (300 x g) remove the supernatant by aspiration being careful not to disrupt cell pellets.

Note: If multiparameter analysis, i.e. surface receptors and proliferation markers, is to be performed, cell "fixation" at this point may not be desirable. Please contact Phoenix Flow Systems, Inc. for further information on their Stick-It^tm and Crisp^tm cell treatment solutions.




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

3. ABSOLUTE-S STAINING PROTOCOL

1. Resuspend each tube of the cell pellets in 50 micro liters of the DNA Labeling Solution prepared as described below.

   DNA LABELING SOLUTION	1 ASSAY	5 ASSAYS	10 ASSAYS
   TdT Reaction Buffer(green cap) TdT Enzyme(yellow cap) Br-dUTP(violet cap) Distilled H2O	10.00 µl 0.75 µl 8.00 µl 32.25 µl	50.00 µl 3.75 µl 40.00 µl 161.25 µl	100.00 µl 7.50 µl 80.00 µl 322.50 µl
   Total Volume	51.00 µl	255.00 µl	510.00 µl

   The appropriate volume of Staining Solution to prepare for a variable number of assays is based upon multiples of the component volumes combined for 1 Assay. Mix only enough DNA Labeling Solution to complete the number of assays prepared per session. The DNA Labeling Solution is active for approximately 24 hours.

2. Incubate the cells in the DNA Labeling Solution for 60 Minutes at 37C in a temperature controlled water bath. Shake the cells every 15 minutes to resuspend.

3. At the end of the incubation time add 2 ml of Rinse Buffer (red Cap) to each tube and centrifuge each tube (300 x g) for five minutes. Remove the supernatant by aspiration.

4. Resuspend the cells pellet in 0.1 ml of the Antibody Solution (prepared as described below).

   ANTIBODY SOLUTION	1 ASSAY	5 ASSAYS	10 ASSAYS
   Flourescein~PRB-1(orange cap) Rinse Buffer (red cap)	5.00 µl 95.00 µl	25.00 µl 475.00 µl	50.00 µl 950.00 µl
   Total Volume	100.00 µl	500.00 µl	1000.00 µl

5. Incubate the cells with the Fluorescein~PRB-1 Antibody Solution in the dark for 30 minutes at room temperature.
   Hint: Wrap tubes with aluminum foil.

6. Add 0.9 ml of the Propidium Iodide/RNase A Solution (amber bottle) to the tube containing the 0.1 ml Antibody Staining Solution.
   NOTE: If the cell density is low, decrease the amount of PI/RNase A solution to 0.5ml.

7. Incubate the cells in the dark for 30 minutes at room temperature.

8. Analyze the cells in Propidium Iodide/RNase Solution by flow cytometry.

9. Analyze the cells within 3 hours of staining.

 

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