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FACS Protocol Help! - (Jan/11/2007 )

Hi I am a little unsure about my FACS preparation protocol and I need some help confirming if any of my steps are unnecessary.

I need to use the FACS on live cells to determine uptake.

1) Wash with PBS
2) Trypsinize cells
3) Inactivate trypsin with media
4) Pellet cells and resuspend in PBS (x2)
5) Immediately proceed for FACS

I'm wondering if step 3 is really necessary. I've seen some protocols which head straight to step 4. Will the little bit of remaining trypsin affect the viability of the cells?
Also, do I need to wash twice in step 4 (i.e. pellet, resuspend, pellet, resuspend)?

Thanks for your help!

-xtrios-

Actually I have another question to add:

How long can the cells last during the preparation process? I'm using HepG2 cells, and due to the large number of samples, it can take me about 1hr + for the whole trypsinize, wash, pellet, wash procedures. I have kinda assumed the cells are still good and they look fine under the microscope but it is likely that they are actually dieing in that time?

-xtrios-

QUOTE (xtrios @ Jan 11 2007, 10:05 AM)
Hi I am a little unsure about my FACS preparation protocol and I need some help confirming if any of my steps are unnecessary.

I need to use the FACS on live cells to determine uptake.

1) Wash with PBS
2) Trypsinize cells
3) Inactivate trypsin with media
4) Pellet cells and resuspend in PBS (x2)
5) Immediately proceed for FACS

I'm wondering if step 3 is really necessary. I've seen some protocols which head straight to step 4. Will the little bit of remaining trypsin affect the viability of the cells?
Also, do I need to wash twice in step 4 (i.e. pellet, resuspend, pellet, resuspend)?

Thanks for your help!


there are discussions if it is necessary to inactivate trypsin by medium; for some reasons, it depends on your trypsin; for recombinant trypsin like Gibcos TrypLE there appears to be no need for inactivation which, however, does not imply long-term incubation; washing in PBS removes both medium and trypsin; medium components, especially if using FCS, may disturb staining process for FACS analysis

-The Bearer-

Thanks for the quick reply!

The trypsin I'm using is actually from Sigma. I have tried skipping the media inactivation step the last few runs and I did not notice any drop in the quality of results (I did save a lot of time). Intuitively, I thought the washing should remove most of the trypsin in the solution anyway, especially after two washes. Just wanted to make sure I wasn't doing anything that would ruin the validity of my results.

-xtrios-

hi,

you should try citric saline since trypsin damage some proteins at the cell surface
http://www.protocol-online.org/forums/inde...howtopic=17456

Sebastien

-tryptofan-

I used to inactivate trypsin and add PBS (10x fold vol. ) and proceed to FACS directly.

-scolix-

Thanks for the citric saline protocol. I will probably use this in place of trypsin. Just need to check a couple of things:

Do I need any inactivation with citric saline?
Will the citric saline affect fluorophores binding to cell surface?

Thanks!

-xtrios-

QUOTE (tryptofan @ Jan 11 2007, 12:06 PM)
hi,

you should try citric saline since trypsin damage some proteins at the cell surface
http://www.protocol-online.org/forums/inde...howtopic=17456

Sebastien


sorry, canĀ“t open the link; but the thing seems to be that citric acid (3 acid groups) chelates Ca2+; so, some use only EDTA w/o trypsin; I suppose that even EGTA would work better than EDTA (each 4 acid groups)

-The Bearer-

QUOTE (xtrios @ Jan 11 2007, 11:57 AM)
Thanks for the quick reply!

I have tried skipping the media inactivation step the last few runs and I did not notice any drop in the quality of results (I did save a lot of time). Intuitively, I thought the washing should remove most of the trypsin in the solution anyway, especially after two washes. Just wanted to make sure I wasn't doing anything that would ruin the validity of my results.


I agree with you, 2 washes should be suficient to remove trysin. But I guess all depends how quick you are in processing all the samples. If you have many samples and you cannot use cell sorter immediatly, I guess having some active trysin might eventually start to disrupt cell membrane, or not?
I know that you can use trypsin to digestion membrane cell wall and one don't want to make artificially membrane more permeable because that would affect PI stainning results.

Because I have a lot of samples and always take like an hour to collect everything and have them ready I do the extra step of trypsin inactivation with FCS.

Good luck

-macedo-

HepG2s don't like being without FBS very much, but 1-2h will be fine if you don't want to recover the cells after doing FACS. Important is also that you'll have to sqeeze the HepG2 cells (if it is the hepatocarcinoma cell line) through a needle several times to avoid clogging the FACS nozzle. Probably also need to do it fairly regularly during antibody staining or you'll get uneven staining.



QUOTE (xtrios @ Jan 11 2007, 08:14 AM)
Actually I have another question to add:

How long can the cells last during the preparation process? I'm using HepG2 cells, and due to the large number of samples, it can take me about 1hr + for the whole trypsinize, wash, pellet, wash procedures. I have kinda assumed the cells are still good and they look fine under the microscope but it is likely that they are actually dieing in that time?

-LeserattePD-