Why two mountains occur in dcfda assay through facs ?
my protocol is following
1. detached cell using TE and drug treatment in HBSS media.
2. after appointed time (1,2 h), dcfda staining during 30 min in 37℃ and dark.
3. measurement
Regards,
Daiha Shin
Hi Daiha,
Your fluorescence histograms are in Log Scale.
On the second histogram you have a population identifdied by a peak with 10^1 AU fluorescence, and another population with 10^2 AU. This means the second piopulation is 10-fold brighter than the first.
In other words this means that the first population has a basal metabolic fluorescence of ten, and the second has this basal fluorescence + DCF fluorescence. First population is negative (dead), second is positive (alive).
If you can you can also stain your cells with 1µg/mL Propidium Iodide, and see if DCF negative cells are also PI positive.
Cheers
Dear Olivier Catrice,
Thanks for your discussion, I got it. However, I wonder if only cells alive are measured. If there is, I want to know that.
Best regards,
DiCabrboxyFluoresceinDiAcetate (DCF-DA) is an hydrophobic dye. It can then cross plasma membrane freely.
Once in the cytoplasm, cytosolic esterases hydrolyze acetate groups and DiCarboxyFluorescein (DCF) is released, which is highly hydrophilic. It cannot thus cross any membrane and is retained is the cytosol, which appears brightly green.
If cells are DCF positive, this means they have both active esterases, so they have an active metabolism, AND they have intact plasma membrane, otherwise DCF would not be retained in the cytosol.
You can assume with very few mistake that cells with active metabolism and intact plasma membrane are alive.
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