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

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