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Help for cell preparation for FACS...


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#1 CaiJLe

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Posted 01 November 2010 - 06:15 PM

I do FACS for GFP expressing cells WITHOUT fixation.(well, my tutor advised not to fix.)


FIRST, I have to sort cells from TISSUE not plated cells so trypsinization to depart each cell is matter. Usual method used in my lab is 15min, 37C with trypsin.
This method seems to give quite much loss. I once tried with trypsin-EDTA but I failed to adjust proper time and temperature.

Anyway, when I run FACS, there are too many(actually, almost..)dead cells.(forward scatter value less then 50.) I use retinal cells which are quite small but such a small value shows cells are dead.

In addition, GFP signal is so weak. Quenching of GFP due to pH was the point my tutor said. Do you know any other reason?
(should I check intensity of signal after fixation to use like control?)

#2 Rsm

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Posted 02 November 2010 - 08:51 AM

I do FACS for GFP expressing cells WITHOUT fixation.(well, my tutor advised not to fix.)


FIRST, I have to sort cells from TISSUE not plated cells so trypsinization to depart each cell is matter. Usual method used in my lab is 15min, 37C with trypsin.
This method seems to give quite much loss. I once tried with trypsin-EDTA but I failed to adjust proper time and temperature.

Anyway, when I run FACS, there are too many(actually, almost..)dead cells.(forward scatter value less then 50.) I use retinal cells which are quite small but such a small value shows cells are dead.

In addition, GFP signal is so weak. Quenching of GFP due to pH was the point my tutor said. Do you know any other reason?
(should I check intensity of signal after fixation to use like control?)


Very good, no fixation. I don't understand why people would fix GFP expressing cells? Makes no sense to me...
I would recommend using Dispase/Collagenase instead of Trypsin for solid tissues. That is much more gentle, cuts only extracellular stuff, no dead cells. Your FSC values also indicate lots of cell debris. You will have much less with Disp/Coll.
No GFP signal can have many reasons, the major one being no or low expression. eGFP is quite pH stable. I would recommend using a positive control, like cells where you GFP is expressed (maybe somewhere in the blood lineage?), and compare this to a negative sample. Your sample should then be somewhere in between.

rsm
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