This material was originally published in the Purdue Cytometry CD-ROM Series,volume 4


Marco Vitale*, Giorgio Zauli° and Elisabetta Falcieri+

*Dept. Biomedical Sciences and Biotechnologies, University of Brescia, Italy °Institute of Human Anatomy, University of Ferrara, Italy +Institute of Anatomy and Physiology, University of Urbino, Italy.




1. Introduction

 It may be useful to make some general comments on the flow cytometric distinction of apoptotic from necrotic cells before describing the methods.

 2A. First protocol: light scatter

2A.1. Materials
 -PBS, pH 7.0
 -Flow cytometer with forward (FSC) and side (90°) scatter (SSC) detection, laser tuned at 488nm wavelength

2A.2. Methodology


3A.  Commentary

3A.1. Background information

3A.2. Time considerations

3A.3.  Key references

2B. Second protocol: membrane permeability by propidium iodide

2B.1. Materials

2B.2. Methodology

3B. Commentary

3B.1. Background information

3B.2. Troubleshooting

3B.3. Time considerations

3B.4. Key references

 2C. Third protocol: membrane permeability by Hoechst/PI

2C.1. Materials

2C.2. Methodology

3C. Commentary

3C.1. Background information

3C.2. Time considerations

3C.3. Key references

2D. Fourth Protocol: DNA content

2D.1. Materials

2D.2. Methodology

3D. Commentary

3D.1. Background information

3D.2. Time considerations

3D.3. Key references


1. Introduction 

2. Protocol

2.1. Materials

2.2. Methodology


  1.  At least 500.000 cells or a 1 mm x 1 mm tissue fragment are necessary to have a specimen proper for ultrastructural analysis.
  2. The glutaraldehyde fixation must be performed immediately, possibly preceeded by a PBS washing, to remove culture or biological medium. During fixation, cells must not be touched, to avoid resuspension, which could cause cell loss.
  3. After 1 h at RT, a washing is required, with 0.15 M phosphate buffer, which keeps the physiological osmolarity, to prevent shape changes or distortions. The permanence in buffer can last until 8-10 days, at 4°C, eventually changing the buffer with a new fresh one.
  4. Postfixation is carried out in 1% OsO4 in the same buffer, for 1 h RT, followed by a rapid wash in the same buffer.
  5. CAUTION: Both glutaraldehyde and OsO4 fixation cause vapour exhalations and manipulations must be carried out with care and under chemical hood.
  6. Specimen dehydration follows by means alcohol-water solutions of increasing concentrations (50%, 70%, 95%, 100%, 15-20 min each) and final treatment with 100% propylene oxide for 30 min. Araldite embedding starts with a 1:1 mixture of propylene oxide:araldite for 1 h, followed by 1:3 mixture overnight, at RT.
  7. After an additional treatment in undiluted resin for 1 h, polymerization is performed at 60°C for 3-4 days.
  8. Sectioning is generally preceeded by the analysis of semithin sections, stained at 40°C with 1% toluidine blue, which gives an overall information about the specimen, thus facilitating the choice of thin section areas.
  9. Staining of thin sections, collected on nickel or copper grids, is performed by means of uranyl acetate and lead citrate for 10 and 15 min, respectively. Careful washing is necessary between the two treatments, as well as at the end of the procedure.

Observations with conventional transmission electron microscope are generally performed at 80 KV.


  1. Smaller cell amonts can be usefully processed for this ultrastructural approach. In the case of isolated cells, they must be fixed in suspension (not as a pellet, as for TEM) with the same glutaraldehyde as above, after having carefully removed the medium, with a gentle centrifugation before fixation.
  2. For SEM, cell adhesion to a substrate is required and it is obtained by cell suspension deposition on poly-L-lysine coated slides and storing at 4°C, in a moist chamber, overnight.
  3. Slides are therefore postfixed and dehydrated as above. A crucial step for SEM procedure is solvent removal in the absence of specimen distortions or drying artifacts. This is now performed by a critical point drying device, which utilizes the progressive substitution of the solvent with liquid CO2, successively evaporated by high pressure-heating.
  4. Finally, specimens are mounted on sliver paint-coated conventional stubs, and sputtered with gold to stabilize their surface and prevent electric charge effects. Observations with conventional scanning electron microscope are generally performed at 1-10 KV, depending on specimen characteristics and coating quality.


  1. Specimens, fixed with 2.5% glutaraldeyde as above, and washed, are mounted on gold supports and cryoprotected with 30% glycerol in 0.1 M phosphate buffer pH 7.3, for 1-3 h, to prevent ice crystal formation, which could disrupt membranes and organelles.
  2. Subsequently, they are quickly frozen in Freon 22 and rapidly transferred into liquid nitrogen. The fracture is performed at -115°C and platinum-carbon/carbon replicas of the exposed surfaces are obtained, checking the thickness with a quartz film monitor. Organic tissue deletion is obtained by cleaning with commercial bleach and degreasing with a chloroform-methanol mixture.
  3. Purified replicas are gently washed with distilled water and collected on nickel grids, previously coated with formvar and carbon.

3. Commentary

3.1. Background information

3.2. Critical Parameters

3.3. Troubleshooting

3.4.  Time considerations

3.5. Key References

  1. Falcieri, E., Zamai, L., Santi, S., Cinti, C., Gobbi, P., Bosco, D., Cataldi, A., Betts, C., and Vitale, M. 1994. The behaviour of nuclear domains in the course of apoptosis. Histochemistry 102: F221.
  2. Falcieri, E., Mariani, A. R., Mariani, E., Gobbi, P., Facchini, A., and Manzoli, F. A. 1990. A morphological study of membrane lesions during natural killer-mediated lysis. J. Submicrosc. Cytol. Pathol. 22: F191.
  3. Stuppia, L., Gobbi, P., Zamai, L., Palka, G., Vitale, M., and Falcieri, E. 1996. Morphometric and functional study of apoptotic cell chromatin. Cell Death Diff. 3: F397.
  4. Falcieri, E., Gobbi, P., Cataldi, A., Zamai,  L., Faenza,  I., and Vitale, M. 1994. Nuclear pores in the apoptotic cell. Histochem. J. 26: F754.