Problems with fixation? - (Mar/19/2012 )

Hi,

I write to ask you for some suggestions. I explain you my problem: I use to fix my cells and then to perform an antibody staining. The fixation protocol involves the next steps:

- PFA 4%, 15min at room temp
- Triton 0.5% 5min at room temp
- (after 2 washing steps) BSA 0.4% 30min at room temp

My problem is that the FSC signal I usually get from my samples is not homogeneous at all, the signal can be really different from one sample to the other one (even 100% difference), and this reflects also on the signal coming from the antibody.

I guess this problem could be due to some bugs in the fixation, but I'm not that expert at all.

Do you have some useful clues/ tips?

Thank you in advance!

Baloo

FSC is a measurement for the cell size, so I guess your fixation step results in cell clumping. 4% PFA for 15mins is a rather long fixation, can you maybe decrease that time (5min should be enough) or use different fixation (like EtOH)? Also, is it really necessary to fix your cells? Many antibodies have problems with fixed antigens, and you will get cell aggregates.

Hi Post Dog,

thanks for answering. Unfortunately I need to fix my cells. Perhaps I could try to reduce the incubation time, but if aggregates are produced I should be able to see them if I look at my cells at the microscope, isn't it? Sometimes I look at them just to check that the staining worked, and in fact it seems to work fairly well, and I don't see a lot of aggregates.
What do you think about this?

Thanks

P.S.
Just for information, I use to centrifuge my cells (CHO) at 200g.

Have you ever tried the Cytofix/Cytoperm reagent (BD) ? I use it to permeabilize and fix my cells before flow cytometry.

Tabaluga on Mon Mar 19 19:02:13 2012 said:

This!

I use the intracellular staining buffers (that are part of the foxp3 kit, but I order them separately- I think they are essentially the same as the cytofix/cytoperm) and I love them.
Before using them I had so many problems with my flow, but now nothing.

Honestly, do yourself a favour and just buy the stuff from BD!
The extra expense will be well worth it compared to your time and energy in making the reagents you currently use (esp. the PFA, jeez I hate making that!!), and the time you are wasting trying to optimise your fixation.

Thanks for your answer. I think now I will first try to reduce the incubation time for PFA, and then perhaps I will have a look at the products you suggested me. Of course, it would be nice and faster if I can solve the problem just incubating in PFA for a shorter time!

Hi Baloo,
a collegue in our lab just discovered the same problem! he found out that if you fix your cells with ethanol, the FSC signal is MUCH more stable! the only problem is, that you must check if then your antibody staining still works fine.