Introduction
 

Cells undergoing apoptosis display typical features, namely cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. Dramatic changes occurr within the nucleus during apoptotic death. It is commonly thought that the nuclear changes are due to activation of endogenous nuclease(s) which cleaves DNA into oligonucleosomal fragments. This is associated with the appearance of dense, crescent-shaped chromatin aggregates which line nuclear membrane. Later, the nucleulus disintegrates, nuclear membrane develops deep invaginations and, ultimately, the nucleus fragments into dense granular particles (apoptotic bodies).
Chromatin condensation, nuclear shrinkage and formation of apoptotic bodies can easily be observed under fluorescence microscopy, after appropriate staining of nuclei with DNA-specific fluorochromes. Both adherent cell lines and cells growing as suspension can be examined. However, due to slight differences in the fixation procedures, two separate methods will be described. A short paragraph dealing with fixation requirements for confocal analysis is also included.

Materials

• PBS pH 7.2 (stock solution 2X)
• Paraformaldehyde 3.7% (A1)
• Absolute Methanol (or Ethanol)

• dissolve 3.7 g of paraformaldehyde in 50 ml H₂O, add 2 drops of 10N NaOH, heat 30 min at 60°C. Add 50 ml of PBS 2X. Adjust pH to 7.2
• The PFA solution must be freshly made.
• Attention PFA is toxic if inhaled.

1. Wash cells in PBS pH 7.2
2. Fix cells in 3.7% paraformaldehyde (PFA) in PBS pH 7.2 for 15 min. at room temperature
3. Wash once with PBS for 5 min at room temperature
4. Cover the cells with methanol 5 min. room temperature
5. Wash cells in PBS pH 7.2
6. Incubate with staining solution

• 3.7% PFA and 0.2% TritonX-100 in PBS for 5 min at room temperature
• 3.7% PFA and 0.2% SDS in PBS for 5 min at room temperature
• Methanol for 5 min at room temperature and Acetone for 2 min. at -20°C

 
2. Cells growing in suspension

Materials

• PBS pH 7.2
• Paraformaldehyde 3.7% (A1)
• Absolute Methanol (or Ethanol)

• Cytocentrifuge
• Centrifuge

1. Wash cells in PBS pH 7.2
2. Spin the cells down (2x10⁵ cells 5 min. at 500g)
3. Air dry
4. Restart from point 2 of previous paragraph

 Nuclei can be stained with different fluorochromes, depending on experimental requirements. For example, if a simultaneous analysis of membrane antigens with FITC and/or TRITC is performed, nuclei should be stained with a UV-excited, blue-emitting fluorochrome such as DAPI (excitation 358 nm, emission 461 nm). Propidium iodide (excitation 536 nm, emission 617 nm) can be used for routinary analysis of nuclear morphology or in combination with green-emitting fluorochromes (i.e. FITC).

 
DAPI Staining
 
Materials

• 4’,6-diamidine-2’-phenylindole dihydrochloride (DAPI ) (A1)
• Citric acid
• Tween 20
• Na₂HPO₄

•  Epifluorescence microscope with appropriate filter set

• Dissolve 1mg /ml of powder in water and make appropriate aliquots that can be stored at -20°C

 

                                                 Citric acid 4.2 g (0.2M)
                                                 Tween 20 0.5 ml
                                                 Distilled H₂O 100 ml

                            2) Staining solution

Methodology:

• Resuspend cells in 1 vol of pretreatment solution and incubate 5 min at room temperature
• Add 9 vol of staining solution and incubate 5 min
• Put cells into the slide and observe under fluorescence microscope

Adherent cells:      1) Pretreatment solution

Citric acid 2.1 g
Tween 20 0.5 ml
Distilled H₂O 100 ml

         2) Staining solution

          Citric acid 11.8 g
          DAPI 0.2 mg (200 m l from stock solution)
          Distilled H₂O 100 ml

• Put on cells 1 Vol of pretratment solution and incubate 5 min at room temperature
• Add 6 Vol of staining solution and incubate 5 min
• Remove the excess of liquid and observe under fluorescence microscope

 
Support Protocol ( Short Protocol)

Materials

• DAPI staining solution ( 10 m l from stock solution in 100 ml of PBS)

• Wash once cells with DAPI staining solution for 5 min at room temperature and observe directly under fluorescence microscope (this short method is suitable for nuclear counter-staining in immunofluorescence analysis)

Propidium Iodide Staining

 
Materials

• Propidium Iodide Stock solution 1 mg/ml in PBS (A1)
• PBS pH 7.2

• Epifluorescence microscope with appropriate filter set

• Dissolve 1mg /ml of PI powder in PBS.

 Suspension Cells

• Resuspend cells in 1 ml PBS
• Add 10 ml PI stock solution
• Incubate 10 min at room temperature
• Examine under the fluorescence microscope

• Cover cells with the appropriate volume of PI working solution (10 mg/ml PI in PBS).
• Wash with PBS and mount.
• Observe under the microscope

Protocol for Confocal Fluorescence Microscopy: Fixation

Confocal laser scanning microscopy (CLSM) has recently emerged as a technique which offers several advantages over conventional fluorescence microscopy. The out-of-focus blur is virtually absent from confocal images, giving the capability for serial optical sectioning of intact specimens and subsequent three-dimensional reconstruction. For a precise analysis of stereo-spatial relationships of different cellular organelles, it is critical that fixation and mounting of specimens preserve the structural architecture of the cells. The ideal fixative should penetrate tissues quickly, act rapidly and preserve the cellular structure before the cell can react to produce structural artifacts. The fixation methods described below have been optimized for CLSM analysis of apoptotic cells and nuclei.
 

1. Cells growing in suspension

Materials

• PBS pH 7.2
• Paraformaldehyde 3.7% in CSK (A1)
• 0.2% TritonX-100
• Poly-L-lysine stock solution (mw 300000, 0.01% in water) (A2)

•  Dakopen
•  Coverslips

• Dissolve PFA as above, add 10 ml CSK 10X.
• Adjust pH to 7.4
• 10X CSK: 100 mM Pipes pH 6.8 (adjust with 10N NaOH)
• 1 M NaCl
• 30 mM MgCl₂
• 20 mM EDTA
• 3 M sucrose

* The PFA solution with sucrose should be made freshly
 

(A2) Preparation of poly-L-lysine coated coverslips:

• Wash with soap
• Rinse with distilled H₂O
• Immerse coverslip in absolute ethanol
• Immerse coverslip in acetone
• Air dry
• Make a ring with DAKO-pen around the coverslip edge
• Put on the poly-L-lysine solution for 30 min at room temperature
• Pour off exceeding poly-L-lysine
• Wash with distilled H₂O
• Air dry

Methodology

• Centrifuge cells at 1200 rpm for 5min.
• Wash freshly harvested cells (3-5 X 10⁵) in PBS
• Resuspend cell pellet in 300 m l of PBS
• Put on poly-L-lysine coated coverslip 30 min at 4°C (A2)
• Remove (gently!!) unbound cells
• Wash once with PBS for 5 min at room temperature
• Fix in 3.7% PFA in CSK for 10 min at room temperature
• Wash three times in CSK w/o sucrose for 5 min at room temperature
• Add 0.2% Triton X-100 in CSK w/o sucrose for 5 min at room temperature
• Wash once in PBS
• Incubate with staining solution

 2. Adherent cell lines

Materials

• PBS pH 7.2
• Paraformaldehyde 3.7% in CSK (A1)
• 0.2% TritonX-100

• Coverglass

• Growth cells on coverglass and start from point 7 of previous paragraph

Protocol for Confocal Fluorescence Microscopy: Staining

As reported in the previous chapter, in this case too nuclei can be stained with different fluorochromes, depending on experimental demands. Since the majority of CLSMs are equipped with argon (488 nm excitation line) or argon-kripton (488, 568 and 647 nm excitation lines) only fluorochromes with appropriate excitation spectra can be used. Propidium iodide or long-wavelenght nuclear fluorochromes (TO-PRO-3, TOTO-3 from Molecular Probes) are both suitable for revealing nuclear alterations during apoptosis. The staining method is the same reported in the previous paragraph. After staining samples should be properly mounted for CLSM processing:

• Air dry the sample
• Apply four posts of nail polish (to avoid that coverslip touch the sample)
• Apply the appropriate volume of mounting medium (A1) (about 10-20 ml, depending on the height of the specimen)
• Seal the coverslip with nailpolish
• Observe under CLSM

• Make a solution of 20 g polyvinyl alcohol of average mw 10000 (Gelvatol, Monsanto Chemical Co or Airvol 205, Air Products Ltd) in 80 ml of 140 mM NaCl, 10 mM Sodium Phosphate buffer, pH 7.2 by stirring for 16 h or heating in a boiling waterbath
• Add 40 ml glycerol and str for a further 16 h, then centrifuge at 12000 to remove any undissolved solid. Remove the supernatant, check its pH is between 6 and 7 and store in 10 ml aliquots in the freezer (-20°C)
• Add 1 mg DABCO (Sigma) to a 10 ml working solution of this preparation before use and store at 4°

Protocol for Acridine Orange Staining after DNA denaturation  

Acridine Orange (AO) is a metachromatic dye which differentially stains double-stranded (ds) and single-stranded (ss) nucleic acids. When AO intercalates into dsDNA it emits green fluorescence upon excitation at 480-490 nm. On the contrary, it emits red when interacts with ssDNA or RNA. Chromatin condensation is an early event of apoptosis and the condensed chromatin is much more sensitive to DNA denaturation than normal chromatin. Therefore, if RNA is removed by pre-incubation with RNase A and DNA is denaturated in situ by exposure to HCl shortly before AO staining, apoptotic cells (which have a larger fraction of DNA in the denaturated form) display an intense red fluorescence and a reduced green emission when compared to non-apoptotic interphase cells.
 

Materials  

• Acridine Orange (A1)
• Citric acid
• Na₂HPO₄
• Paraformaldehyde
• PBS
• DNAse-free RNAse A (A2)
• Ethanol
• HCl

• Epifluorescence microscope or Flow cytometer

• Acridine Orange 6 mg/ml
• Citric acid 0.1 M
• Na₂HPO₄ 0.2 M pH 2.6
• Prepare 90 ml of citric acid solution, add acridine orange and 10 ml Na₂HPO₄

 
(A2) RNAse solution

• Dissolve 1 mg of RNAse A (use DNAse-free RNase) in 1 ml of distilled water

  Methodology:

1. Wash cells (1x10⁶) in PBS and centrifuge at 200 g for 5 min
2. Resuspend the cell pellet in 1 ml PBS
3. Fix cells by transferring the cell suspension in 9 ml 1% paraformaldehyde in PBS, on ice. Incubate for 15 min on ice
4. Centrifuge at 200 g for 5 min and resuspend the cell pellet in 5 ml PBS, centrifuge
5. Suspend the cell pellet in 1 ml PBS and transfer the suspension in 9 ml 70% (vol/vol) ethanol, on ice.
6. Incubate for 4 h (The cells can be stored in ethanol for weeks)
7. Centrifuge at 200 g for 5 min and resuspend the cell pellet in 1 ml PBS
8. Add 0.2 ml of RNAse A solution. Incubate at 37°C for 30 min
9. Centrifuge at 200 g for 5 min and resuspend the cell pellet in 0.2 ml PBS
10. Add 0.5 ml of 0.1 M HCl at room temperature
11. After 30-45 sec add 2 ml AO staining solution
12. Observe the cells under fluorescence microscope with an appropriate filter set.
13. Alternatively, analyse cells by flow cytometry (excitation 488 nm; dot plot of green fluorescence at 530± 20 nm versus red fluorescence >600 nm).

Key References

1. Hotz MA, F Traganos and Z Darzynkiewicz. 1992.Changes in nuclear chromatin related to apoptosis or necrosis induced by the DNA topoisomerase II inhibitor fostriecin in MOLT-4 and HL-60 cells are revealed by altered DNA sensitivity to denaturation. Exp Cell Res 201:184-191

2. Gorman A, J McCarthy, D Finucane, W Reville and T Cotter. 1994. Morphological assessement of apoptosis. In: Techniques in Apoptosis. A user’s guide. TG Cotter and SJ Martin eds, Portland Press, New York, pp. 1-20

3. Pawley JB.1995. Handbook of Biological Confocal Microscopy (2^nd Edition). Plenum Publishing Corporation, New York.