Acridine Orange Staining Procedure

Acridine Orange (AO) is a nucleic acid selective metachromatic stain useful for cell cycle determination. AO interacts with DNA and RNA by intercalation or electrostatic attraction respectively. DNA intercalated AO fluoresces green (525nm); RNA electrostatically bound AO fluoresces red (>630nm). It may distinguish between quiescent and activated, proliferating cells, and may also allow differential detection of multiple G1 compartments1. AO may also be useful as a method for measuring apoptosis, and for detecting intracellular pH gradients and the measurement of proton-pump activity2.

This procedure optimally stains cells for analysis by flow cytometry.

Stock reagents

0.1M Citric Acid
(Citric Acid, anhydrous (FW 192.1); 1.921g per 100ml dH2O)

0.2M Dibasic Sodium Phosphate
(Sodium Phosphate, dibasic anhydrous (FW 142.0) 2.839g per 100ml dH2O)

Triton X-100 (Baker)
0.5M EDTA, disodium salt (SIGMA) NaCl (FW 58.44)
Acridine Orange (Polysciences or SIGMA) Sucrose, anhydrous (FW 342.3; SIGMA)

Stock Buffer I

20mM Citrate-Phosphate, pH 3.0, 0.1mM EDTA, 0.2M Sucrose, 0.1% Triton X-100
(To 125ml dH2O add 40μl 0.5M EDTA, 26.48ml 0.1M Citric Acid, 6.85ml 0.2M Dibasic Sodium Phosphate, 13.69g Sucrose, 0.2ml Triton X-100. QS to 200ml and 0.2μ filter. Store at 4oC)

Stock Buffer II

10mM Citrate-Phosphate, pH 3.8, 0.1M NaCl
(To 150ml dH2O add 9.92ml 0.1M Citric Acid, 5.46ml 0.2M Dibasic Sodium Phosphate, 1.7g NaCl. QS to 200ml and 0.2m filter. Store at 4oC)

Staining Cells

  1. Make a 2mg/ml AO in dH2O solution – dilute to 1:100 in Buffer II
  2. Aliquot cells: 105 – 106 in 100μl PBS or media
  3. Add Buffer I (0.5ml) at room temp, agitate to suspend
  4. Add Buffer II + AO (0.5ml) at room temp, agitate to suspend
  5. Run on flow cytometer. (Setup will require some red-green compensation to optimize G1 %c.v.)

1 Darzynkiewicz Z. Differential staining of DNA and RNA in intact cells and isolated cell nuclei with acridine orange. Methods in Cell Biology. 33:285-98, 1990
2 Darzynkiewicz Z. Bruno S. Del Bino G. Gorczyca W. Hotz MA. Lassota P. Traganos F. Features of apoptotic cells measured by flow cytometry. Cytometry. 13(8):795-808, 1992

Translate »