Hello, Meaghan Roy-O'Reilly,

I hope (if i am not wrong) you are asking about the cells fixation time, that for how much time you can store thembefore analysis. It has been mentioned in some protocols that we can store the fixed cells at 4°C for weeks, when the cells tubes are tightly closed. However personally i don't believe this. I have got good results after fixing the cells for 30-90 minutes at 4°C. Fixation for long time may lead to excessive cell death giving you false +ve or -ve results.  

Hello Meaghan,

It is totally depend on what test u want to carry? As some tests are affected with time such as (ROS), while others not. And I agree with Salman, when I have fresh sample I have better result than that stored in 4C for long time.

Good luck

What do you fix your cells with? I fix primary cells in 4% PFA straight after isolation, for 10 minutes. Then I wash them in PBS and resuspend in PBS/BSA. I usually stain the next day for FACS; I never leave for more than 2 days at 4 degrees otherwise the cells disintegrate. 

Hope this helps!

That really depends on what you are aiming for. If you are trying to stain for surface markers, I would suggest to run the fresh samples as fixation will increase the autofluorescence in the cells and it will become difficult to differentiate between positive and negative populations. If you are trying to do intracellular staining, you cannot skip the fixation step, it's mandatory. However, you can try to search for surface markers of the your population of interest.

I understand that you are wondering for how long you can store fixed and stained samples before analysing. If that is so then I have done many PBMCs samples and stored them overnight without problem. I had fioxed them with PFA.

Hi Meaghan,

You can fix PBMC's and keep them safely at 4C (I have not tried it with PMNs) for several days. But you have to be aware that not only the cells will be slightly affected, but also some fluorochromes might lose a bit of brightness. As Sarah Boyle says it is important to wash cells after fixation and store them in PBS/BSA or PBS/FBS , without the fixative.

If you are staining major PBMC populations (T-cell, B-cell, monocytes...) it should be fine. If you attempt to identify very tiny subpopulations or markers with a very low expression , I would definitevely try to read on FACS fresh.... but I know it is almost impossible with clinical samples!

Good luck

All of these are great answers.

One big thing to look out for is the epitope binding of the antibody post-fixation. Many antibodies are checked for their ability to work post-fixation for Buffered Formalin, when explicitly stated (i.e good for IHC or other applications on the datasheet), but not all are. Keeping this in mind your fixation technique can also influence the sensitivity of detection too (i.e. MeOH versus 4% Paraform). You may want to do a few mock panels with fresh normal buffy coats to get a good feel on what will work and what will not. As Victor stated above, robust population won’t change much usually the Staining Index typically goes down (the difference between +ve and –ve populations),  but this can be counteracted with appropriate FMO controls (Fluor Minus One). I’ve done this several times and sometimes it works out well where you even get better staining for antigens post-fixation.

All the luck

I agree with John. If you get the chance (and specifically for surface staining), have a go with your Abs on a less important sample such as a buffy coat, both before and after fixation to have a look first (or see whether there is information on the datasheets).

It really depends on what epitopes the individual Abs are recognising and whether the chemical crosslinking with the PFA is affecting access of the Abs to their epitopes. This is even more important if you're using monoclonals which will only recognise 1 epitope. While staining with many Abs is unaffected by this type of fixation, there are also examples where a PFA fixation step can hide the epitope altogether and therefore give you very reduced signals.

If the Ab binding is unaffected by PFA fixation, then you're fine and you should be able to store the samples for at least a few days (though they do slowly degrade, even with fixation so I wouldn't go much longer than that). If it is affected, then you may have to consider use of a different Ab clone/reagent or performing all the stains prior to fixation. As standard, we stain then fix, but this may not be possible with clinical samples.

Good luck!

Thank you for all of the answers! After reading, I think we'll try letting them sit at 4*C overnight at the very most. We're getting samples from acutely injured patients at all hours of the day, so fixation first looks like it may be a necessary evil. We've experimented with fix/stain and stain/fix and it looks like our antibody recognition isn't significantly diminished (we're looking at large, robust cell populations for now, thankfully!). We can cryopreserve our PBMCs, but I know our PMNs won't survive the freeze, so we're going to try and run as many "fresh fixed" samples as we possibly can. This has been very helpful.

Hi Meaghan,

I just saw your question and wanted to add my '2 cents' and this particular point was not mentioned in the previous responses.  Be very careful with tandem dyes.  Some, such as PerCP-Cy5.5, are ok if the sample is fixed for several days.  Others, such as PE-Cy5, will begin to degrade after 24hrs, even with fixation.  When that happens, in a tube stained for just the PE-Cy5 ab, you will see neg, PE-Cy5+ and PE+ signals.  This is due to the Cy5 molecule 'falling off' the PE molecule.  You can imagine that running PE-Cy5 and PE conjugated ab's in the same tube can cause no end of confusion in your results.  Good luck.

You mentioned above that you fix and then stain, and at the end of the question you say: how many days could that be left before staining and flow?. In my case i always stain before fixing, never after. I try to find appointments for flow for the same day , or asap, but if it is too crowded, i have been able to read 7 days after staining and still get the same results than when I read the same day or the day after. Just as a cautionary measure, keep your stained samples covered in foil, preferably in a dark,  not very popular place at 4oC until the time of your appointment. Good luck!

Sorry, Meaghan:

I have similar question. I would ask Irina one more question. Do you mean the fixation does not affect the Ab that already bind on the cells. If this works well, I would be very happy to follow because I always have 10 to 15 animal samples simutaneously, which I really can not handle well at the same time on the same day. Thank you both, Meaghan and Irina.

Sorry Hou , I just read this. Yes. try with one sample. Fix it and Stain it, and go and read it the same day , and 3 days later , and a week later , and you will see that not much changes. Do it so you have an idea of how much your tissue can last ok after fixing and staining.

Thanks a lot, Irina. I appreciate it.

Staining will depend on antibody and antigen only. So what happens to them by whatever procedure you use must be tested under conditions you will expect to use.

I prefer to use ethanol to fix cells over the use of formaldehyde because of possibilities that the formaldehyde will change the antigen epitope sufficiently to loose binding. I use formaldehyde for EM work because I have to (EtOH does not work). I study epithelial cells and cytosolic/mitochondrial proteins (NOT surface proteins) and I have had a great deal of success using ANDREWS GUIDE TO FACS’ing with ethanol and storage (up to weeks)with no problems

https://sciencetechblog.wordpress.com/flow-cytometry-users-guide/

Hello.

Even I had the same doubt,if we can store the fixed sample for few days.As everyone has recommended and have good experience.I will also follow up your guidance for flow cytometry,and update with results.

Thanks.

Hi everyone,

I have a similar problem. When staining cells at room temperature, the receptor is internalized if staining before fixing in 0.5%PFA. I am trying to fix before staining, but see an increase in signal in my negative control and a decrease in my positive control. Any suggestions? The assay cannot be performed cold.

Thanks!

Always stain first. Fixing alters protein structure impacting antibody binding

Hi

I am working with bacterial cells and I from a mixed population I need to stain and sort my target organism Fam probe and also determine its viability with PI. Should I stain before I fix or vice versa , any info will be appreciated.

Thanks