EMS10050

Materials:

  • PBS pH 7.2 (stock solution 2X)

  • Paraformaldehyde 3.7% (A1)

  • Absolute Methanol (or Ethanol)

(A1) Preparation of paraformaldehyde (PFA) solution (100ml):

  • Dissolve 3.7g of paraformaldehyde in 50ml H2O, then add 2 drops of 10N NaOH, heat 30 minutes at 60°C. Add 50ml of PBS 2X. Adjust the pH to 7.2

  • The PFA solution must be freshly made

NOTE: PFA is toxic if inhaled

Staining Procedure:

  1. Wash cells in PBS pH 7.2

  2. Fix cells in 3.7% paraformaldehyde (PFA) in PBS pH 7.2 for 15 minutes at room temperature

  3. Wash once with PBS for 5 minutes at room temperature

  4. Cover the cells with methanol for 5 minutes at room temperature

  5. Wash cells in PBS of pH 7.2

  6. Incubate with staining solution

Protocol 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 releases green fluorescence upon excitation at 480-490nm. It emits red, however, upon interacting with ssDNA or RNA.

Chromatin condensation is seen in the early stage of apoptosis and the condensed chromatin is significantly more sensitive to DNA denaturation than is normal chromatin. If RNA is removed by pre-incubation with RNase A and DNA is denaturated in situ by exposure to HCl immediately before AO staining, apoptotic cells display a strong 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

Equipment:

  • Epifluorescence Microscope or Flow Cytometer

(A1) Staining solution

  • Acridine Orange 6 µg/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

Acridine Orange solution is stable for several weeks when it is stored at 4°C as well as in the dark

(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 200g for 5 minutes

  2. Re-suspend the cell pellet in 1 ml PBS

  3. Fix cells by transferring the cell suspension in 9ml 1% paraformaldehyde in PBS, on ice. Incubate for 15 minutes on ice

  4. Centrifuge at 200g for 5 minutes and re-suspend the cell pellet in 5ml PBS, centrifuge

  5. Suspend the cell pellet in 1ml PBS and transfer the suspension in 9ml 70% (vol/vol) ethanol, on ice

  6. Incubate for 4 hours (These cells may be stored in ethanol for weeks at a time)

  7. Centrifuge at 200g for 5 minutes and re-suspend the cell pellet in 1ml PBS

  8. Add 0.2ml of RNAse A solution. Incubate at 37°C for 30 minutes

  9. Centrifuge at 200 g for 5 minutes and re-suspend the cell pellet in 0.2ml PBS

  10. Add 0.5ml of 0.1M HCl at room temperature

  11. After 30-45 seconds add 2ml of AO staining solution

  12. Observe the cells under fluorescence microscope with an appropriate filter set

  13. You may also analyse cells by cytometry (excitation 488nm; dot plot of green fluorescence at 530 ±20nm versus red fluorescence >600nm)