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Should I fix cells digested from tissues? - (Apr/26/2011 )

I am digesting animal tissues(with collagenase I) to look for cells that were injected systemically, to find where they engraft. The cells were pre-labelled with a red fluorescent dye (taken up into the lipid bilayer of the membrane). Since there will be a lot of differnt organs at each timepoint, I won't be able to get through all the digests and flow cytometry on the same day. So, I was going to fix the single cell suspensions in paraformaldehyde, and then look for labelled cells, stain for other markers and analyse by flow over the following days to make it more manageable. I am not sure whether I need to stain first, and then fix, and which fixation protocol would be best. Would fixation in 4% paraformaldehyde made up in PBS for 10 mins be a suitable approach? Following fixation, should the cells be resuspended in PBS, and how long will they remain suitable for analysis? Since the tissue harvest and digestion will be very time consuming, the minimum time for preparation on day 1 would be ideal, then the following days can be devoted as necessary. My very limited experience with flow has been on fresh cell samples. I would be delighted to receive any advice/suggested protocols for this.

Many thanks

-phoebe75-

I am not a big supporter of fixation for any flow appliction. Fix always results in loss of epitopes and cell clumps. How long is your ColI treatment? Usually, you will have a good single cell suspension after 1-2h (at least when using ColI/Dispase at 3mg/ml each). I would suggest you do either a long day in the lab (ie. do the flow directly after this digestion), or digest overnight (but then may cells may die). Best way is to try different conditions and fnd the most suitable way.

-Rsm-

We used 1% para instead of 4%. It was a softer fixation and seemed to stain better. The length of time you can hold the cells depends on the number of colors--a single-color is good for a few weeks, a double, a week, three-color for a few days...etc. Keep them at 4C in the dark and they should be OK.

-lab rat-

Rsm on Wed Apr 27 07:56:05 2011 said:


I am not a big supporter of fixation for any flow appliction. Fix always results in loss of epitopes and cell clumps. How long is your ColI treatment? Usually, you will have a good single cell suspension after 1-2h (at least when using ColI/Dispase at 3mg/ml each). I would suggest you do either a long day in the lab (ie. do the flow directly after this digestion), or digest overnight (but then may cells may die). Best way is to try different conditions and fnd the most suitable way.

Thanks for responding- based on what I have read, I agree it would be best to try to avoid fixation. It's just becasue of the numbers involved and the length of time it takes to do the harvesting that we need to continue into the next day- will just get the whole tissues in late evening.

-phoebe75-

lab rat on Wed Apr 27 08:35:49 2011 said:


We used 1% para instead of 4%. It was a softer fixation and seemed to stain better. The length of time you can hold the cells depends on the number of colors--a single-color is good for a few weeks, a double, a week, three-color for a few days...etc. Keep them at 4C in the dark and they should be OK.

Thanks for that information- very helpful. We will try 1% parafromaldehyde and store at 4C in the dark as you said. Thanks again!

-phoebe75-