Contents: APO-DIRECT™
Catalog Number: 88-6611
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Description
The APO-DIRECT™ Kit is a 2-color staining method for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry. The kit contains the instructions and reagents required for measuring apoptosis in cells including positive and negative control cells for assessing reagent performance; washing, reaction and rinsing buffers for processing individual steps in the assay; terminal deoxynucleotidyl transferase enzyme (TdT), Fluorescein-deoxyuridine triphosphate and propidium iodide/RNase A solution for counterstaining the total DNA.
One of the most easily measured features of apoptotic cells is the break-up of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result in the appearance of DNA laddering when the DNA is analyzed by agarose gel electrophoresis. The DNA of non-apoptotic cells that remains largely intact does not display this laddering on agarose gels during electrophoresis. The large number of DNA fragments appearing in apoptotic cells results in a multitude of 3'-hydroxyl termini in the DNA. This property can be used to identify apoptotic cells by labeling the 3'-hydroxyl ends with directly conjugated fluorescein- deoxyuridine triphosphate nucleotides (FITC-dUTP). The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes a template-independent addition of deoxyribonucleoside triphosphates to the 3'-hydroxyl ends of double- or single-stranded DNA with either blunt, recessed or overhanging ends. A substantial number of these sites are available in apoptotic cells providing the basis for the method utilized in the APO-DIRECT™ Kit. Non-apoptotic cells do not incorporate significant amounts of the FITC-dUTP due to the lack of exposed 3'-hydroxyl DNA ends.
Sufficient
reagents are provided to process 50 cell suspensions including 5 ml positive and 5ml negative control cell suspensions of approximately 1 x 106 cells per ml in 70%(v/v) ethanol.
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APO-DIRECTTM Protocol
The following protocol describes the method for measuring apoptosis in the positive and negative control cells that are provided in the APO-DIRECTTM Kit. The same procedure should be employed for measuring apoptosis in the cell specimens provided by the researcher.
- Resuspend the positive (brown cap) and negative (natural cap) control cells by swirling the vials. Remove 1 ml aliquots of the control cell suspensions (approximately 1 x 106 cells per 1 ml) and place in 12 x 75 mm flow cytometry centrifuge tubes. Centrifuge (300xg) the control cell suspensions for 5 minutes and remove the 70% (v/v) ethanol by aspiration being careful to not disturb the cell pellet.
- Resuspend each tube of control cells with 1 ml of Wash Buffer (blue cap) for each tube. Centrifuge as before and remove the supernatant by aspiration.
- Repeat the Wash Buffer treatment (step 2).
- Resuspend each tube of the control cell pellets in 50 µl of the Staining Solution (prepared as described below).
| STAINING SOLUTION COMPONENT |
1 ASSAY |
6 ASSAYS
(2 controls + 4 unknown) |
12 ASSAYS
(2 controls + 10 unknown) |
TdT Reaction Buffer (green cap)
TdT Enzyme (yellow cap)
Fluorescein-dUTP (orange cap)
Distilled H2O |
10.00 µl
0.75 µl
8.00 µl
32.00 µl |
60.0 µl
4.5 µl
48.0 µl
192.0 µl |
120.0 µl
9.0 µl
96.0 µl
384.0 µl |
| Total Volume |
50.75 µl |
304.5 µl |
609.0 µl |
Note: 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.
- Incubate the cells in the Staining Solution for 60 minutes at 37°C in a temperature-controlled bath. Shake cells every 15 minutes to re-suspend.
Note: The Staining Reaction can also be carried out at 22-24°C overnight for the control cells. For samples other than the control cells provided in the kit, incubation times at 37°C may need to be adjusted to longer periods of time.
- At the end of the incubation time, add 1.0 ml of Rinse Buffer (red cap) to each tube and centrifuge each tube (300xg) for five minutes. Remove the supernatant by aspiration.
- Repeat the cell rinsing (as in step 6) with 1.0 ml of the Rinse Buffer (red cap), centrifuge and remove the supernatant by aspiration.
- Resuspend the cell pellet in 0.5 ml of the Propidium Iodide/RNase A solution (amber bottle).
- Incubate the cells in the dark for 30 minutes at room temperature.
- Analyze the cells in Propidium Iodide/RNase Solution by flow cytometry.
- Analyze the cells within 3 hours of staining.
Cell Fixation Procedure for APO-DIRECTTM Assay
Note: Cell fixation using paraformaldehyde is a required step in APO-DIRECTTM assay. The following cell fixation procedure is a suggested method. Variables such as cell origin and growth conditions can affect the results. The fixation conditions provided below should be considered as guidelines. Additional experimentation may be required to obtain results comparable to the control cells provided with this kit. The positive and negative control cells provided in APO-DIRECTTM Kit are already fixed.
- Suspend 1-2 x 106 cells in 0.5 ml of 10 mM sodium phosphate pH 7.2, 150 mM sodium chloride (PBS).
- Add the cell suspension into 5 ml of 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes.
- Centrifuge cells for 5 minutes at 300xg and discard the supernatant.
- Wash the cells in 5 ml of PBS then pellet the cells by centrifugation. Repeat the wash and centrifugation.
- Resuspend the cells in the residual PBS in the tube by gently vortexing the tube.
- Adjust the cell concentration to 1-2 x 106 cells/ml in 70% (v/v) ice cold ethanol. Store the cells in the freezer or on ice for a minimum of 30 minutes before staining. In some biological systems storage of the cells at minus 20°C in 70% (v/v) ethanol for at least 12-18 hours prior to staining for apoptosis detection yields better results.
- Store cells in 70% (v/v) ethanol at minus 20°C until use. Cells can be stored at minus 20°C several days before use.
APO-DIRECT is a trademark of Phoenix Flow Systems, San Diego, California.
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