10 replies to this topic

[FACS_flow]

Hello crew,

Until now I always harvested my cells using a 1% Trypsin/EDTA solution for around 5 minutes in 37°C.
Every once a while I also use cell scrapers if after trypsination cells are still adherent to the flask.

Just recently a colleague told me that this procedure might affect the surface receptors, in which
I am especially interested.
So my question now is, which methods of harvesting adherent cells do you use or propose for me
so that cell surface molecules will not get affected.

Thanks in advance!

[bob1]

Any method will affect the cell surface markers... there isn't really any way around it unless you can fix in the flask and then remove the cells.  Otherwise try a suspension culture.

[tonix37]

see the link UpCell

http://www.nuncbrand...e.aspx?ID=11850

I hope you serve

tonix37

[Neurite]

Hey FACS,

I work with a membrane receptor, and we always use versene      When I do flow citometry, no exception.

http://www.bio-medic...itrogen-3923-1/

The receptors mantain intact....

NEVER use trypsin, it is like a razor for membrane receptors.

Edited by Neurite, 06 May 2009 - 02:25 PM.

[awadh]

For cell surface receptor study it always advisable to avoid trypsinization of cell (detached cells by scraping cells) or use suspension culture for this purpose. But you can use some suspension cultured cells which can be adhered by differentiation, in this case it will be easy to detach cells during experiment. In my case i am using THP-1 cells differentiated by PMA these cells can be simply detached by pipetting up & down 2-4 times.

Neurite, on May 7 2009, 04:52 AM, said:

Hey FACS,

I work with a membrane receptor, and we always use versene      When I do flow citometry, no exception.

http://www.bio-medic...itrogen-3923-1/

The receptors mantain intact....

NEVER use trypsin, it is like a razor for membrane receptors.

[drB]

bob1, on Apr 30 2009, 05:39 PM, said:

Any method will affect the cell surface markers... there isn't really any way around it unless you can fix in the flask and then remove the cells.  Otherwise try a suspension culture.

This is true but some are more gentile than others in maintaining surface marker integrity.  Try 5mM EDTA in PBS (without Ca or Mg).  The Trick here I learned from the Sigma sales man, is to place the cells for 5min on ice, and then place them into a incubator for 10-15min.  They ususally detach quite well.

[Dcienmeder]

I have met the similar problem. Some use "non-enzymatic cell dissociation buffer"(sigma) or directly use cell scraper. I don't know whether these methods work.

[lovenergy]

we just use an angled glass pipette to wash the cells (HepG2) off (physical force), the cells worked fine for later receptor binding assays.

[chiranjit0086]

I have started using FACS recently for analyzing apoptosis. but i faced some problem with the machine while i was doing it. can i keep the PI stained cells for next day until the machine repairs. how long can i keep the stained cells or what should i do if i face some similar problem after staining. plz suggest me...

Thank you
CG

[keefec]

chiranjit0086, on 28 October 2011 - 05:48 AM, said:

I have started using FACS recently for analyzing apoptosis. but i faced some problem with the machine while i was doing it. can i keep the PI stained cells for next day until the machine repairs. how long can i keep the stained cells or what should i do if i face some similar problem after staining. plz suggest me...

Thank you
CG

You can keep until the next day in the 4 degree fridge if you have fixed the cells. But you will probably have bad CVs during analysis.

Personally for PI-EtOH stains, I can keep the cells for a few days without much signal loss. Ultimately, depends on your own cells.

[Hamed Karimian]

I have one idea for you, but its depend on your experiment, if you using anexin v assay, you do not need to do it by flowcytometry, you can use by florescence microscope which you need to have chamberslide, here you can observe the cells by flourcsence microscope whith out disturbing the cell surface...