The University of Iowa Carver College of Medicine

Flow Cytometry Facility

Acridine Orange for
    Cell Cycle Analysis

Reagents

  • Citrate Phosphate Buffers
  • X = 2.1gm citric acid monohydrate in 100ml dH2O (0.1M)
  • Y = 2.84gm anhydrous, dibasic sodium phosphate in 100ml dH2O (0.2M)
  • ph 3.0 buffer 79.45ml of X + 20.55ml of Y (0.04M PO4)
  • ph 3.8 buffer 64.5ml of X + 35.5 ml of Y (0.07M PO4)
  • Stock Buffer #1 (for 100ml)
  • 100µl Triton X-100 (0.1%)
  • 6.85gm sucrose (0.2M)
  • 3.58mg disodium EDTA (10-4M)
  • 50ml pH 3.0 citrate phosphate buffer (M based on phosphate) (2 x 10-2M)
  • QS to 100ml with dH2O
  • Final pH should be about pH3.5
  • Stock Buffer #2 (for 100ml)
  • 0.58gm NaCl (0.1M)
  • 14.25ml pH 3.8 citrate phosphate buffer (1 x 10-2M)
  • QS to 100ml with dH2O
  • Store refrigerated
  • Acridine Orange
  • Stock Solution:  2 mg/ml in dH2O (store refrigerated protected from light)
  • Staining Solution: 0.1ml AO stock solution + 9.9ml buffer #2 (1:100 dilution) (20µg/ml final concentration)
    • Make staining solution (AO + buffer #2) solution fresh for each run
    • Use only AO from Polysciences cat# 4539

Staining

Acridine Histogram
  1. Add 0.5ml buffer #1 to 105 - 106 cells (in 100µl media)
  2. Incubate 1 minute
  3. Add 0.5ml AO staining solution
  4. Run immediately on flow cytometer