MORPHOLOGICAL ASPECTS OF APOPTOSIS
Walter Malorni, Stefano Fais1, and Carla Fiorentini
 
Laboratory of Ultrastructures and (1) Virology, Istituto Superiore di Sanita’,
viale Regina Elena 299, 00161, Rome, Italy.
E-mail: Malorni@ul.net.iss.it

 



Introduction


A) Light microscopy
 
A1. Introduction

A2 Protocol

 

a) Cells Growing In Suspension

 

Aa.2.1 Materials

Polylysine-coated coverslips

 

. Sonicate the coverslips (12 mm) in acetone for 15 min at room temperature.

. Wash the coverslips with distilled water (dH2O) to remove completely the acetone.

. Place the coverslips on a clean paper sheet separing each others. Leave the coverslips dry at air.

. Gently spread 20 ml polylysine solution (0.1 mg/ml dH2O) on the coverslips surface.

. Leave the polylysine drops on the coverslips dry at room temperature.

 

Aa.2.2. Methodology

 

1. Centrifuge cells (2-5 x105) at 1000 rpm for 5 min.

2. Remove the supernatant and resuspend the pellet in 5 ml 3.7% paraformaldehyde in PBS. Leave the sample at room temperature for 15 min.

3. Centrifuge cells at 1000 rpm for 5 min.

4. Remove the supernatant and resuspend the pellet in 5 ml PBS. Centrifuge sample at 1000 rpm for 5 min.

5. Resuspend the pellet in 20-30 m l PBS (depending on the number of cells) and deposit it on polylysine-coated coverslip. Leave the cells adhere on coverslip for 30 min at room temperature.

6. Wash the coverslip twice with PBS.

7. Gently add 0.5% Triton X-100 and leave for 5 min at room temperature. Rinse three times with PBS.

8. Incubate the coverslip with Hoechst 1 mg/ml for 30 min at 37°C.

9. Rinse the coverslip with PBS and mount on the slide with glycerol-PBS (1:1).

10. Observe the sample by a fluorescence microscope (U.V. light).

 

• Slides can be kept in the dark at +4°C for several days.

 

b) Cells growing in monolayer: detached cells (i.e., following apoptotic treatments).

 

Ab.2.1. Materials

 

Ab.2.2. Methodology

 

1. Collect the medium, centrifuge at 1500 rpm for 10 min and wash once with PBS. Resuspend the pellet in 4 ml 3.7% paraformaldehyde in PBS. Leave the sample at room temperature for 20 min.

2. Centrifuge the detached cells at 1500 rpm for 10 min. Wash once with PBS. Centrifuge sample at 1500 rpm for 10 min.

3. Resuspend the pellet in 20 ml PBS and deposit it on polylysine-coated coverslip. Leave the cells adhere on coverslip for 30 min at room temperature.

4. Wash the coverslip twice with PBS.

5. Gently add 0.1% Triton X-100 and leave for 5 min at room temperature. Rinse three times with PBS.

6. Incubate the coverslip with Hoechst 1 mg/ml for 30 min at 37°C.

7. Rinse the coverslip with PBS and mount on the slide with glycerol-PBS (1:1).

8. Observe the sample with a fluorescence microscope (U.V. light).

 

• Slides can be kept in the dark at +4°C for several days.

 

c) Cells growing on coverslips

(or remaining adhering to the substrate).

 

Ac.2.1. Materials

 Sterilization of coverslips (12 mm)
  2. Wash the coverslips with dH2O to remove completely the acetone.

3. Place the coverslips on a clean paper sheet separing each others. Leave the coverslips dry on air.

4. Put the coverslips into a heat-resistant glass bottle.

5. Sterilize the coverslips in autoclave for 15 min at 121¡C (heat-resistant glass bottle must remain partially opened during sterilization).

6. After sterilization, open the heat-resistant glass bottle in the sterile cup. Leave the coverslips dry with U.V. ray.

7. Close the heat-resistant glass bottle and leave it in the sterile cup.

 

Ac.2.2. Methodology

 

1. Remove the medium and wash the cells twice with PBS.

2. Directly add to the sample the 3.7% paraformaldehyde in PBS. Leave for 20 min at room temperature.

3. Remove the fixative and rinse three times with PBS.

4. Directly add to the sample O.5% Triton X-100 and leave for 5 min at room temperature. Rinse three times with PBS.

5. Incubate the coverslip with Hoechst 1 mg/ml for 30 min at 37°C.

6. Rinse the sample with PBS and mount on the slide with glycerol-PBS (1:1).

7. Observe the sample with a fluorescence microscope (U.V. light).

 

• Slides can be kept in the dark at +4°C for several days.

 

Samples can also be stained with propidium iodide (fixing cells with ethanol 70% at +4°C for 30 min) or acridine orange 0.1% (fixing the cells with ethanol) in citrate buffer (pH 3).

 
B) In situ immunocytochemistry


 

 
B1.Introduction

B2.Protocol B2.1.Materials  

B2.2.Methodology

 

*Cytospin preparation:

• Centrifuge 20-80x104 cells/slide at 400-600 rpm (cytocentrifuge Shandon) for 5 min.

• Carefully wipe slide around the cell spot and dry at room temperature .

• Fix the slide sample in absolute acetone for 10 min at 4°C.

• Wash with Tris buffer Solution (TBS)(pH 7.6) twice

• Dry and saturate the sample with 10% Rabbit serum or FCS or AB human serum as appropriate.

• Cover with Tunel solution and incubate for 60 min at 37°C humid atmosphere.

• Wash with TBS twice.

• Dry and cover with the mouse anti-FITC Ab and incubate for 60 min at room temperature in dark condition.

• Wash twice with TBS.

• Dry, cover with linking antibody (usually a Rabbit anti-mouse for both the PAP and APAAP method) and incubate for 30 min at room temperature.

• Wash twice with TBS.

• Dry, cover with APAAP or PAP as appropriate (for Tunel staining we prefer APAAP allowing the use of a wider panel of chromogens, particularly dark chromogen as NBT or fast blue) and incubate for 45 min at room temperature.

• Wash twice with TBS.

• Dry, cover specimens with developing solution with the substrate-chromogen standard composition suitable for either PAP (0.05% 3,3-diaminobenzidine tetrahydrochloride color reaction, DAB) or APAAP (Fast blue or BCIP/NBT for the dark blue staining, Fast red for red staining + N,N-dimethyl-formamide) and incubate 10 to 30 min.

• Block reaction with TBS for 10 min.

• Coverslip with aqueous mounting media (Aquamount).

 

 

*Chamber-slide preparation:

To obtain the best cell morphology the use of chamber slides is recommended. Of course in this case the amount of Tunel mixture as well as of the other reagents highly exceeded that used for cytospin preparation (roughly 5 folds more for chamber slides). Therefore, we use this technique precisely when we have some problem in identifying some morphological parameters. The method is roughly the same of the cytospin preparation, with the only exception of the number of cells used, the technique to attach non-adherent cells on L-polylysin-covered chamber-slide and the permeabilization procedure (unfortunately acetone cannot be used with plastic materials):

• place 2-5x105 cells in each chamber in the case of adherent cells. In the case of non-adherent cells prepare poly-L- covered chamber slides (fill with poly-L-lysyn 1:100 in PBS each chamber; incubate at room temperature for 30 min; wash, place 5x105/chamber and incubate for 2 h at 37°C in humid atmosphere; wash and fix).

• treat each chamber with methanol 70% or ethanol 80% for 10 min at +4°C.

• wash and follow the same procedure as in (a).

 

*Cryostat sections:

The technique is roughly the same described in (a) with the only exception of the preliminary procedures for collecting, cutting and treating tissue specimens. In particular:

• collect tissue sample, place the specimen in OCT and immediately snap freeze on liquid nitrogen.

• place at -80°C.

• cut 4-5 m sections using a -25°C cryostat.

• air-dry for 2h to overnight

• wash with PBS1x to remove OCT

• dry and fix with 100% cold acetone for 10 min at +4°C

• wash and air-dry.

• carefully wipe slide around the section

• follow the same procedure as in (a).
 


C) Scanning Electron Microscopy (SEM)

 

 
C1. Introduction
 

C2. Protocol

 

a) Cells growing in suspension

 

Ca.2.1. Materials
 

C.a.2.2. Methodology
 

Fixation

 

1. Centrifuge cells (2-5 x 105) at 1000 rpm for 5 min.

2. Remove the supernatant and resuspend the pellet in 5 ml 0.1M cacodylate buffer. Centrifuge sample at 1000 rpm for 5 min.

3. Remove the supernatant and resuspend the pellet in 5 ml 2.5% glutaraldehyde in 0.1M cacodylate buffer. Leave the cells fix for 20 min at room temperature.

4. Centrifuge cells at 1000 rpm for 5 min.

5. Remove the supernatant and wash the cells twice with 5 ml 0.1M cacodylate buffer.

After fixation with glutaraldehyde, samples can be kept at +4°C in 0.1M cacodylate buffer for several days.

6. Centrifuge cells at 1000 rpm for 5 min.

7. Resuspend the pellet in 20-30 ml 0.1M cacodylate buffer and deposit it on polylysine-coated coverslip. Leave the cells adhere on coverslip for 20 min at room temperature.

8. Delicately add 0.1M cacodylate buffer.

Remove 0.1M cacodylate buffer and add 1% osmium tetroxide (OsO4) in 0.2M cacodylate buffer. Leave the cells fix for 30 min at room temperature.

9. Wash the coverslip three times with 0.2M cacodylate buffer. After post-fixation with OsO4, samples can be kept at +4°C in 0.2M cacodylate buffer for 1-2 days.

 

Dehydration through graded ethanols and critical point dried (CPD)

 

10. Remove the 0.2M cacodylate buffer. Add 30% ethanol. Leave for 5 min and remove (repeat three times).

11. Remove the 30% ethanol. Add 50% ethanol. Leave for 5 min and remove (repeat three times).

12. Remove the 50% ethanol. Add 70% ethanol. Leave for 10 min and remove (repeat twice). At this point the dehydration procedure may be stopped for up to one week. Samples can be kept in 70% ethanol at +4°C.

13. Remove the 70% ethanol. Add 85% ethanol. Leave for 15 min and remove (repeat twice).

14. Remove the 85% ethanol. Add 95% ethanol. Leave for 15 min and remove (repeat twice).

15. Remove the 95% ethanol. Add absolute ethanol. Leave for 10 min. Repeat three times leaving the samples always covered by ethanol during preparation of CPD. Never leave the samples dry.

16. Dehydrate samples by CPD.

17. Glue the coverslips onto stubs with silver print. Let dry about 20 min at room temperature and overnight at 37°C.

18. Cover the samples with gold (thickness 20 nm) using the sputter coater.

 

• Conserve the samples at 37°C in a speciment dessiccator.

 

b) Cell lines which grow in monolayer

(on the coverslips)

 

C.b.2.1. Materials

 

1. coverslips

2. 0.1M cacodylate buffer (pH 7.4)

3. 2.5% glutaraldehyde in 0.1M cacodylate buffer

4. 0.2M cacodylate buffer (pH 7.4)

5. 1% osmium tetroxide (OsO4) in 0.2M cacodylate buffer

6. Graded ethanols

7. Stubs

8. Silver print

 

C.b.2.2. Methodology

 

1. Remove the medium and wash the cells twice with 0.1M cacodylate buffer.

2. Directly add to the sample the 2.5% glutaraldehyde in 0.1M cacodylate buffer. Leave the cells fix for 20 min at room temperature.

3. Remove the fixative and rinse three times with 0.1M cacodylate buffer.

After fixation with glutaraldehyde, samples can be kept at +4°C in 0.1M cacodylate buffer for several days.

4. Remove 0.1M cacodylate buffer and add 1% osmium tetroxide (OsO4) in 0.2M cacodylate buffer. Leave the cells fix for 30 min at room temperature.

 

 

To note: When cells are adhering on plastic flasks (and not on coverslips), small plastic fragments can be easily obtained (e.g. with shears) and processed for SEM by step N.9

 

 

D) Transmission Electron Microscopy (TEM)


 
D1. Introduction

 
 

D2.Protocol

 

 

D2.1. Materials

 

 

D2.2. Methodology

 

Fixation:

 

*Dispersed cells

 

1. Centrifuge cells (2-5 x 106) at 1000 rpm for 5 min.

2. Remove the supernatant and resuspend the pellet in 5 ml 0.1M cacodylate buffer. Centrifuge sample at 1000 rpm for 5 min.

3. Remove the supernatant and resuspend the pellet in 5 ml 2.5% glutaraldehyde in 0.1M cacodylate buffer. Leave the cells fix for 20 min at room temperature.

4. Centrifuge cells at 1000 rpm for 5 min.

5. Remove the supernatant and wash the cells twice with 5 ml 0.1M cacodylate buffer.

After fixation with glutaraldehyde, samples can be kept at +4°C in 0.1M cacodylate buffer for several days.

6. Centrifuge cells at 1000 rpm for 5 min.

7. Remove the supernatant and resuspend the pellet in 3-4 ml 1% osmium tetroxide (OsO4) in 0.2M cacodylate buffer. Leave the cells fix for 1h at room temperature.

8. Centrifuge cells at 1000 rpm for 5 min.

9. Remove the supernatant and wash the cells twice with 5 ml 0.2M cacodylate buffer.

After post-fixation with OsO4, samples can be kept at +4°C in 0.2M cacodylate buffer for 1-2 days.

10. Centrifuge cells at 1000 rpm for 5 min.

11. Remove the supernatant and embed the pellet in 2% agar.

12. Cut the embedded pellet in pieces of about 1mm3. Put the pieces in a small glass bottle.

 

 

*Cell lines growing in monolayer

 

1. Remove the medium and wash the cells twice with 0.1M cacodylate buffer.

2. Directly add to the sample the 2.5% glutaraldehyde in 0.1M cacodylate buffer. Leave the cells fix for 20 min at room temperature.

3. Remove the fixative and rinse twice with 0.1M cacodylate buffer. After fixation with glutaraldehyde ,samples can be kept at +4°C in 0.1M cacodylate buffer for several days.

4. Remove 0.1M cacodylate buffer and add 1% osmium tetroxide (OsO4) in 0.2M cacodylate buffer. Leave the cells fix for 1h at room temperature.

5. Remove the fixative and add 0.2M cacodylate buffer. Leave for 5 min at room temperature and remove (repeat three times). After post-fixation with OsO4, samples can be kept at +4°C in 0.2M cacodylate buffer for 1-2 days.

 

*Animal tissues

 

1. Cut the tissue in pieces of about 1mm3, leaving the samples always covered by 0.1M cacodylate buffer. Never leave the samples dry.

2. Put the tissue pieces in a small glass bottle always covered by 0.1M cacodylate buffer.

3. Remove the buffer and directly add 2.5% glutaraldehyde in 0.1M cacodylate buffer completely covering the tissue pieces. Leave the cells fix for 2h at +4°C.

4. Remove the fixative and add 0.1M cacodylate buffer. Leave for 30 min at +4°C and remove (repeat twice).

5. Remove the buffer and directly add 1% osmium tetroxide (OsO4) in 0.2M cacodylate buffer completely covering the tissue pieces. Leave the cells post-fix for 30 1h at +4°C.

6. Remove the fixative and add 0.2M cacodylate buffer. Leave for 5-10 min at room temperature and remove (repeat three times). After post-fixation with OsO4, samples can be kept at +4°C in 0.2M cacodylate buffer for 1-2 days.

 

Dehydration through graded ethanols

 Dehydrate the samples through graded ethanols as follows:

Prepare a sature solution of uranyl acetate. Remove 70% ethanol and, after filtration, add this solution to the samples. Leave for 30 min at room temperature. All these procedures must be conducted in the dark. Then continue as follows:

 
 
70% ethanol  2 x 10 min  2 x 5 min  2 x 10 min
 

  At this point the dehydration procedure may be stopped. Samples can be kept in 70% ethanol at +4°C.

 
85% ethanol 1 x 10 min 1 x 10 min  1 x 10 min 
95% ethanol  1 x 30 min 1 x 10 min  1 x 30 min
Absolute ethanol 3 x 20 min 3 x 10 min 2 x 30 min
Propylene oxide 
(or toluene)
1 x 10 min   1 x 20-30 min 
 

Inclusion

Include the samples as follows:
 
dispersed cells tissues
Propylene oxide/Resin 2:1 1 x 1h 1 x 1h
Propylene oxide/Resin 1:1 1 x 1h 1 x 1h
Propylene oxide/Resin 1:2  1 x 2h 1 x 2h
At this point the inclusion procedure is usually stopped for an overnight. Samples can be kept in Propylene oxide/Resin 1:2 at +4°C.

 
Absolute resin 2 x 1h 1 x 2h
Absolute resin into the beems 1 x 1h 1 x 2h
 
 
 
 
Monolayer
Absolute ethanol/Resin 2:1 1 x 1h
Absolute ethanol/Resin 1:1 1 x 1h
Absolute ethanol/Resin 1:2 1 x 1h
 

At this point the inclusion procedure may be stopped for an overnight. Samples can be kept in absolute ethanol/Resin 1:2 at +4°C.

 
Absolute resin 2 x 1h
Absolute resin with the beems  1 x 1h
 

Monolayers: fill beems with absolute resin and put them upside down on the cell layer.
 

Polymerization

Monolayers: gently detach the beems. They will detach with small fragments of polymerized resin. A dark surface will indicate the presence of cells.

• Polymerize at 60°C for 48h.
 
3. Commentary


 
3.1. Background information

3.2. Troubleshooting 1. The procedures described can be easily performed in cultured cells or in ex vivo experiments on dispersed cells. For analyzing tissues, Tunel and TEM analyses are the preferential techniques.

2. Often the morphological features of apoptosis can be confused or overlap those typical of necrosis. For instance: the so-called secondary necrosis is difficult to be recognized and, in any case, necrosis should also be always evaluated; superaggregation of chromatin could often occur in situations other than apoptosis; lymphocytes very rarely undergo chromatin clumping although undergoing apoptosis; surface blebbing as well as surface smoothing are common markers of necrosis as well as of apoptosis, etc. Moreover, because apoptosis is an asyncronous process, all the apoptotic stages are usually detectable, earlier (light chromatin marginalization) and latest stages (residual bodies).

3. In general, all the in situ methods need years of experience for evaluating data. Some problems may come in quantifying positive cells both in cytospin or polylisine preparations and in cryostat sections, and some counterstaining procedures and/or double staining with cell specific markers may help in better distinguish cells in the various stages of apoptosis. In particular, double staining procedures aimed to discriminate apoptotic to proliferating cells are recommended when an effect on cell differentiation, of a given drug or cytokine, may be in progress.

4. The amount of cells represents a critical factor for studies on apoptosis. For dispersed cell analyses (e.g. lymphocytes) few cells are necessary for light microscopy and SEM studies, about 200.000 cells. For TEM analyses, the pellet should be of at least 5-6x106 cells.

5. Advantages in using light microscopy analysis are represented by the time necessary for performing the assay. On the other hand, the pletora of information provided by SEM and TEM approaches, e.g. the study of organelles and cytoskeleton, is wide, may be useful for subsequent studies, e.g. biochemical or molecular, and furnishes important controls for any experimental tasks.

3.3. Anticipated results.  

3.4. Time considerations

3.5. Key References
  1. Kerr, J.F.R., Wyllie, A.H., and Currie, A.R. 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26: 239-257.

2. Allen, T.D. Ultrastructural aspects of cell death. In: Perspectives on mammalian cell death. (C.S. Potten, ed.) Oxford University Press, Oxford, pp.39-65, 1987

3. Bellomo G., F. Mirabelli, M Vairetti, F. Iosi, and W. Malorni 1994. Morphological and biochemical investigations on plasma membrane blebbing during cell injury. In: In vitro toxicity indicators, Methods in toxicology, part B: (C.A. Tyson and J.M. Frazier eds.), vol 1, pp. 58-71.

4. Malorni, W., Rivabene, R., Maria Teresa Santini, M.T. and Gianfranco Donelli, G. 1993. N-acetylcysteine inhibits apoptosis and decreases viral particles in HIV-chronically infected U937 cells. FEBS Letters, 327, 75-78.

5. Allen, R.T. 1997 Morphological and biochemical characterization and analysis of apoptosis J. Pharmacol. Toxicol. Methods 37, 215-228, 1997

6. Gold, R., Schmied, M., Rothe, G., Zischlier, H., Breitschopf H., Wekerle, H., Lassmann, H. 1993. Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections. J. Histochem. Cytochem. 41: 1023-1030.

7. Negoescu, A., Lorimier, P., Labat-Moleur, F., Drouet, C., Robert, C., Guillermet, C., Brambilla, C. and Brambilla, E. 1996. In situ apoptotic cell labelling by the Tunel method: improvement and evaluation on cell preparations. J. Histochem. Cytochem. 44: 959-968.

8. Fais, S., Burgio, V.L., Silvestri, M., Capobianchi, M.R., Pacchiarotti, A., Pallone, F. 1994. Multinucleated Giant Cells generation induced by IFN-gamma. Changes in the expression and distribution of the intercellular adhesion molecule-1 during macrophage fusion and MGC formation. Lab. Invest: 15: 1033-36.

9. Fais, S., Capobianchi, M.R., Abbate, I., Castilletti, C., Gentile, M., Cordiali Fei, P., Ameglio, F., Dianzani, F. 1995. Unidirectional budding of HIV-1 at the site of cell-to-cell contact is associated with co-polarization of intercellular adhesion molecule-1 and HIV-1 viral matrix protein. AIDS 9: 329-35.
 

Aknowledgments Figures.

 

 

Fig. 1

 


Fig. 2

 


Fig. 3


Fig. 4  


Fig. 5-14

 
 

Fig. 15-22

Fig. 23-27