Acridine orange

Sigma A8097-10 mL

10 mg/mL = 33mM

MW = 301.81

Use at ~20µM

• 1.2 µL added to 2 mL in dish

• incubate 15 min at 37

• change medium

• incubate 15 min at 37

• Replace medium with PBS

• observe with

  • B excite - G emission: dsDNA
  • G excite - R emission: RNA, ssDNA

Got good results initially, but within minutes, cells started to ball up and pull off the substrate. Will try new PBS first, then a concentration gradient of acridine orange.

Old PBS produced fast shrinking and balling up. Newer PBS was less bad. Now try with non-CO2 medium. Does it interfere with fluorescence?

No cell shrinkage in non-CO2 medium. At short exposure times, there is a background glow, but picking the right exposure takes care of that.

This is time sensitive. The red emission (which should be RNA & ssDNA) gradually fades, or overlies the green emmision (which should be ds DNA)

See attached images, taken in order 1, 2, 3. #3 is at about 15 minutes. 1 & 2 are 100x, 3 is 200x.