GFP Detection

From Genome@Yale

Agilent Protocol
Pluronic acid‎

20% pluronic acid (Invitrogen catalog # P-3000MP, 1mL, 30uL aliquots in -20°C)
0.5mM CBNF (carboxynaphthofluorescein diacetate, MW 560.52, Invitrogen catalog # C13196)
  - dissolve 10mg in 1.7mL DMSO for 10mM stock, aliquot, store at -20°C
HBSS (Hanks Balanced Salt Solution)
HBSS + 0.05% pluronic acid (dilute 30uL 20% pluronic acid into 12mL HBSS)

Technical note: prevent cells from clumping
   do not tap or hit the flask during trypsin treatment
   do not over trypinize the cells
   pipet repeatedly to disperse the cells
   proceed immediately to analyze the cells after harvest, staining and washing

1. wash cells with 2mL PBS
2. add 200uL 0.25X Trypsin/EDTA
3. add 1mL media + 10% FBS and transfer the cells to eppendorf tubes
4. spin cells at 400rcf for 3 minutes at room temp
5. resuspend cells in 1mL HBSS + 0.05% pluronic acid
6. add 2uL 0.5uM CBNF
7. incubate 15 minutes at room temperature with gentle mixing (rotator)
8. spin cells at 400rcf for 3 minutes at room temp
9. wash cells with 1mL HBSS + 0.05 pluronic acid
10. spin cells at 400rcf for 3 minutes at room temp
11. carefully remove all liquid
12. resuspend cells in 25uL cell buffer by gentle tapping and mixing
13. analyze the cells on bioanalyzer