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I have just prepared a chapter describing variety of methods to
recognize apoptosis and necrosis by flow cytometry. One of the
methods deals with DNA strand break labeling - single step. To
whoever wants the protocol in a cook-book form, I am enclosing the
text below:
8. Method VII. Fluorochrome labeling of DNA strand breaks in
apoptotic cells.
Activation of the apoptosis-associated endonuclease results in
extensive DNA cleavage and,
thus, generates a large numbers of DNA strand breaks. The presence of
3' hydroxyl termini of
the strand breaks can be detected by attaching to them biotin or
digoxigenin conjugated nucleotides
in a reaction catalyzed by exogenous terminal deoxynucleotidyl
transferase (TdT) [23-25] or DNA
polymerase [25]. Fluorochrome conjugated avidin or digoxygenin
antibodies are used in a second
step of the reaction to label DNA strand breaks. A simplified, single
step procedure, has been
recently developed, utilizing deoxynucleotides directly conjugated to
fluorochromes [26].
Detection of DNA strand breaks requires cell pre-fixation in the
crosslinking agent such as
formaldehyde which, unlike ethanol (Method VI), prevents the
extraction of degraded DNA.
Thus, despite numerous washings that are required for cell staining,
the DNA content of early
apoptotic cells (and with it the number of DNA strand breaks), is not
markedly diminished
compared to unfixed cells. The single step procedure utilizing
BODIPY-conjugated d-dUTP (B-
dUTP) is presented below.
8.1. Reagents
* Prepare fixatives as follows:
1st fixative: 1% methanol-free formaldehyde (available from
Polysciences Inc.,
Warrington, PA, USA) in PBS, pH 7.4.
2nd fixative: 70% ethanol.
* The TdT reaction buffer (5 x concentrated) contains the
following:
potassium (or sodium) cacodylate, 1 M.
Tris-HCl, 125 mM, pH 6.6.
bovine serum albumin (BSA), 1.25 mg/ml
* Cobalt chloride (CoCl2), 10 mM.
* TdT in storage buffer, 25 units in 1 ?l.
The buffer, TdT and CoCl2 are available from Boehringer Mannheim,
Indianapollis, IN.
* BODIPY-16-dUTP (B-dUTP), 50 nmoles in 50 ?l of the storage
buffer [alternatively , use
fluorescein-16-dUTP (f-dUTP)]. Both available from Molecular
Probes, Inc.
* Rinsing buffer.
Dissolve in PBS:
Triton X-100, 0.1% (v/v).
BSA, 5 mg/ml.
* PI Staining buffer.
Dissolve in PBS:
PI, 5 ?g/ml.
DNase-free RNase A, 200 ?g/ml.
8.2. Staining procedure
1. Fix cells in suspension in 1% formaldehyde for 15 min on ice.
2. Centrifuge, resuspend cell pellet in 5 ml of PBS, centrifuge,
resuspend cells
(approximately 106 cells) in 0.5 ml of PBS.
3. Add the above 0.5 ml aliquot of cell suspension into 5 ml of
ice-cold 70% ethanol.
The cells can be stored in ethanol, at -20oC for several weeks.
4. Centrifuge, remove ethanol, resuspend cells in 5 ml of PBS,
centrifuge.
5. Resuspend the pellet (not more than 106 cells) in 50 ?l of a
solution which contains:
10 ?l of the reaction buffer.
0.5 ?l (0.5 ?g) of B-dUTP (or 1 ?g of f-dUTP).
0.5 ?l (12.5 units) of TdT in storage buffer.
5 ?l of CoCl2 solution.
34.8 ?l distilled H2O.
Transfer this cell suspension into 1.5 ml Eppendorf tube.
5. Incubate cells in this solution for 60 min at 37oC
(alternatively, incubation can be carried
on at 22-240C overnight).
6. Add 1.5 ml of the rinsing buffer, centrifuge.
7. Repeat cell rinsing in 1.5 ml of the rinsing buffer, centrifuge.
8. Resuspend the cell pellet in 1 ml of PI staining solution.
9. Incubate 30 min at room temperature in dark.
10. Analyze cells by flow cytometry.
illuminate with blue light (488 nm laser line or BG12
excitation filter)
measure green fluorescence of B-dUTP (or f-dUTP) at 530?20
nm.
measure red fluorescence of PI at >600 nm.
Commercial kits: Phoenix Flow Systems (San Diego, CA, USA) is going
to provide a kit
(ApoDirect TM) to identify apoptotic cells based on a single step
procedure utilizing TdT and B-
dUTP. A description of the method, which is nearly identical to the
above, is included with the
kit. Another kit, based on two-step DNA strand breaks labeling with
digoxygenin-16-dUTP by
TdT, is provided by ONCORTM Inc., (Gaithersburg, MD, USA).
Results: Identification of apoptotic cells is simple due to their
intense labeling with B-dUTP (Fig.
7), which frequently requires use of exponential scale (logarithmic
photomultipliers) for data
acquisition and display (Fig. 7). Simultaneous measurement of DNA
content makes it possible to
identify the cell cycle position of both, cells in apoptotic and
nonapoptotic populations.
8.3. Advantages, limitations
This method appears to be the most specific in terms of positive
identification of apoptotic
cells. Namely, necrotic cells, or cells with primary DNA breaks
caused by X-rays irradiation (up
to the dose of 25 Gy), or DNA damaging drugs, have by an order of
magnitude fewer DNA
strand breaks compared to apoptotic cells [23]. Because cellular DNA
content of both apoptotic
and nonapoptotic cell populations is measured, the method offers a
unique possibility to analyze
the cell cycle position, and/or DNA ploidy, of apoptotic cells. The
method also appears to be
useful for clinical material, in leukemias, lymphomas and solid
tumors [27]. Its major
disadvantage stems from complexity and high cost of the reagents.
Good luck!
Xun Li and Zbigniew Darzynkiewicz
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