Apoptotic Nuclei

Fig. 2. Nuclear changes typical of apoptotic cell death. Murine leukemia cells were incubated alone (top panels) or with 50 (im valinomycin Cbottom panels) for 2 h at 37°C. Cells were stained with Hoechst 33342 and observed by UV microscopy. Normal nuclei have "structure;" variations in fluorescent intensity reflect the distribution of euchromatin and heterochromatin. Apoptotic nuclei, in contrast, have highly condensed chromatin that is uniformly fluorescent. This can take the form of crescents around the periphery of the nucleus, or the entire chromatin can be present as one or a group of featureless, bright spherical beads.

by activated NK cells. This pathway is probably of little consequence in the killing of targets directly recognized by NK cells as it requires much longer to initiate than the perforin/granzyme B pathway. However, it could play a role in killing bystander cells that express functional Fas receptor and should be considered. Potential targets for bystander killing by NK cells in vivo could be activated T and B cells, macrophages, and certain subsets of bone marrow progenitors (23,26).

4. Although the demonstration of extensive DNA fragmentation and a nucleosome ladder have been used as absolute evidence for apoptosis, this conclusion is not warranted as there are now numerous examples of cells that do not degrade their DNA to oligonucleosomes during apoptosis. For example, some cells undergo random double-stranded DNA cleavage only every 50-300 kilobases; this event may actually precede internucleosomal cleavage in most cell types undergoing apoptosis. Nevertheless, the large fragments cannot be detected in conventional sedimentation or agarose gel electrophoresis assays but instead are observed using pulsed-field electrophoresis or by neutral sucrose-gradient density centrifugation (27). Once again, it is important to note that atypical chromatin cleavage was observed using regimens that induced typical apoptosis in related cells; the changes were not observed if the cells were killed via necrosis. Cells that are necrotic may also undergo a minor amount of DNA fragmentation although this is typically not internucleosomal in nature.

5. CAUTION: Acridine orange, ethidium bromide, and propidium iodide have been found by the Ames test to be highly mutagenic and should be handled with care.

6. One practical drawback of the TUNEL technique is that it is much more expensive to perform than other methods (e.g., staining with AO + EB costs approx $0.10 per sample vs as much as $8.00 per sample using TUNEL). It is highly recommended that TdT as well as FITC-dUTP be titrated by the investigator to avoid using them in excessively high concentrations. The wash step with TdT buffer (Subheading 3.5., step 11) optimizes conditions for TdT and is critical for achieving good end labeling with dUTP and TdT at low concentrations.

7. Radiolabeling DNA may lead to high (>30%) values for spontaneous DNA fragmentation which may not be associated with apoptotic morphology. If the cells being tested are infected with mycoplasma, the mycoplasma may incorporate the radiolabel, which will appear in the S or T fractions during quantification, leading to an apparently high spontaneous DNA fragmentation even though the cells appear healthy. In general, high levels of spontaneous DNA fragmentation suggest that the cells are being adversely affected. This finding should not be ignored and adequate measures should be taken to obtain lower values of spontaneous fragmentation and apoptosis.

8. The final ethanol concentration used to permeabilize the cells should be 60-70% as higher concentrations will cause excessive shrinkage and difficulties in obtaining a cell pellet as well as acquiring data in the flow cytometer.

9. For optimal results it is recommended that the cells be stored in 70% ethanol for at least 3 d (Subheading 3.5., step 8) before continuing with end-labeling.

10. In some cells reversing steps 3-8 such that cells are fixed/perme-abilized with 70% ethanol prior to fixation with paraformaldehyde may provide better results.

11. To avoid excessive clumping of cells, it is recommended that paraformaldehyde and ethanol be added slowly as the cells are gently mixed.

References

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12. Kagi, D., Ledermann, B., Burki, K., Zinkernagel, R. M., and Hengartner, H. (1996) Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu. Rev. Immunol. 14, 207-232.

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23. Miyawaki, T., Uehara, T., Nibu, R., Tsuji, T., Yachie, A., Yonehara, S., and Taniguchi, N. (1992) Differential expression of apoptosis-

related Fas antigen on lymphocyte subpopulations in human peripheral blood. J. Immunol. 149, 3753-3758.

24. Leithauser, F., Dhein, J., Mechtersheimer, G., Koretz, K., Bruderlein, S., Henne, C., Schmidt, A., Debatin, K. M., Krammer, P. H., and Moller, P. (1993) Constitutive and induced expression of APO-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells. Lab. Invest. 69, 415^-27.

25. Maciejewski, J., Selleri, C., Anderson, S., and Young, N. S. (1995) Fas antigen expression on CD34+ human marrow cells is induced by interferon gamma and tumor necrosis factor alpha and potentiates cytokine-mediated hematopoietic suppression in vitro. Blood 85, 3183-3190.

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  • Yolanda
    What are apoptotic nuclei?
    2 months ago

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