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.

SYTO RNASelect Green Fluorescent Cell Stain (Invitrogen S32703)

$210 from Invitrogen

Fluoresces green when bound to RNA.

excite: 490 nm, emit: 530 nm

Can be used in live or fixed cells. Fix in methanol, NOT formaldehyde!

Don't use in conjunction with red-orange dyes.

Stock:

100µL 5 mM in DMSO. Store at -20C, dessicated, dark.

To thaw: warm to RT, spin down.

Should be stable for >= 1 year.

Make labeling solution

Make 5µM intermediate stock:

2 µL 5mM stock + 1998 µL medium or PBS

Make 20 100 µL aliquots in 1.5 mL tubes

Label: RNASelect - 100 µL - 5 µM intermediate stock in PBS; Add 900 µL to make labeling solution

Make 500 nM labeling solution in medium or PBS

100 µL 5 µM intermediate + 900 µL medium

Protocol

Live cells

• Cells on coverslip
• Warm 500 nM labeling solution to 37C
• Incubate at 37C 20 min
• Rinse twice in PBS or medium
• Add warm medium, let cells rest 5 min
• Fix in chilled methanol 10 min at -20C
• Several washes in PBS

Fixed cells

• Remove coverslip from medium
• Fix in chilled methanol 10 min at -20C
• Remove methanol, let slip sit in PBS 5 min
• Apply labeling solution 20 min RT
• Wash 5 min in PBS
• Mount coverslip