PFA Fixation strength - Summary

From: Uriel TK <utk@netvision.net.il>
Date: Fri May 02 2008 - 16:42:39 EDT

Hello friends,
thanks a lot for the kind responses. Here's the summary. As always, there were new and important things to learn from the responses. The bottom line for my current purposes is that fixation with PFA is a continuous process and the extent of fixation to be performed depends on the needs, and should be determined empirically. I intend to fix my cells with the different settings that gave good light scatter, put them in the fridge 24 hours, and check them again. It's a natural selection type of experiment: only the fit will survive!

best regards,

Uriel.

-- 
Uriel Trahtemberg, M.D. M.Sc.
PhD student
The Laboratory for Cellular and Molecular Immunology
The Hebrew University - Hadassah Medical Organization
Jerusalem - ISRAEL

I am not a totally useless... at least I serve as a bad example.

PFA is a solid and hydrolyses, rather than dissolves, to give a formaldehyde solution.  In my experience, formaldehyde solutions (whether derived from PFA solid or formalin) don’t give thorough permeabilisation in any useful time period on their own (PI staining shows permeabilisation primarily). 

I think this is a fairly universal experience, leading manufacturers to market “fix and perm” style reagents that both fix and permeabilise.  On lymphocytes and similar cells, you may have only 10 to 15% PI uptake after ½ an hour with 1% formaldehyde, depending on conditions of pH and temperature, leaving overnight you may achieve 85%  or more (depending on temperature).

There are some quite interesting artefacts arising from the partial permeabilisation, mainly that some fluorochromes (CFSE eg) exhibit different spectra (and need different compensation) in cells which are fixed and naturally permeable (and internally fixed by formaldehyde) compared to cells in the same sample which are just surface fixed.

To answer the question on correct fixation may require some re-phrasing, depending on why you’re fixing:  If  (like my ex-colleague) you fix to prevent viral replication, then do a plaque assay; if you’re fixing to prevent some other function of a viable cell, assay that function; if you need to permeabilise a cell and fix its morphology, then use a commercial kit, or devise your own method based on a surface fix and a detergent permeabilisation.

------------------------------------------

        The gold standard is--will they grow.  So wash some up and put them back into culture.

        Second thing to think about--a mitochondrial charge fluorochrome.  Did the mitochrondria loose charge?

        Other possible tests--membrane integrity as in AnxV or merrocyanine 540.  Both should show some change in membrane fluorescence profiles.  Fluorescine diacetate is nonfluorescent until cells cleave the molecule to fluoricine (FITC).  Cells with functional enzyme should turn the cells bright green.  Finally and cheapest--look at them on a phase contrast microscope and compare fixed with fresh cells.  The fixed cells should show a change in phase contrast.

---------------------------------------

You have very nicely presented the questions. “But then, how can I asses if the cells are "really fixed"? How much fixation is enough - what is the "threshold" for fixation with PFA?”

I think the answer depends on what you are trying to do with the fixed cells. For example, if you are trying to detect IL-4 positive cells, you want a sufficient concentration of PFA to immobilize the cytokine and keep it from leaking out. You could assess this my measuring the MFI and percent positives in polyclonally activated T cells. Additionally, you want to use the minimum concentration of PFA required, so that you do not increase non-specific binding, denature the Ag, or do other “bad” things that happen after PFA fixation. Thus, my optimization of PFA fixation entails pretty much doing a titration of concentration using a fixed temperature and then looking at how well cytokines stain.

In contrast, if your endpoint of interest is cell death assessed by PI staining, you would want to use a concentration of PFA that kept the membrane impermeable to PI. Thus, there is no one answer to your question, as the readout of “optimal fixation” depends on what function you are using it for.

BTW, we use 4% PFA warmed to 37 C for 5 minutes (on the bench top). My technician makes up 2 liters of PFA and then aliquots it into 15 ml tunes and freezes the whole lot at –20 C. Once thawed, each tube is tossed after a week.

-----------------------------

I don't know if this will help specifically, but you might have a read
of a book chapter entitled, "Fixation and fixatives" by David Hopwood,
found in "Theory and Practice of Histological Techniques" by John

Bancroft A. Stevens and D.R. Turner (eds.) Churchill Livingstone,  p.21-42, 1990. 

---------------------------------------

The present gold standard for fixation is electron microscope
grade glutaraldehyde together with electron microscopic observation. With
Centrifugal Cytology preparations, one can often see the differences between
PFA and glutaraldehyde  fixation. However, one should be aware that air
drying from an aqueous solution with either fixative can create very
significant artifacts. One simple way to test fixation is to put the cells in
distilled water and subsequently view them with a light microscope. Electronic
cell volume measurements can be employed to optimize the tonicity of fixative
solutions. It should be noted that if the cells are exposed to organic
solvents, their electronic cell volume distribution will be altered, since the
membrane will no longer be a resistor and their light scattering will also be 
altered because of a change in refractive index and/or size.. 

-------------------------------------

The question should really be revised to ask whether the cells are
fixed enough for what you want to do, because the fixation is
(presumably) a means to an end, not an end in and of itself.

For intracellular cytokine staining (for instance), simply titrate your
fixation conditions against stimulated T cells using one set of
previously determined optimal stimulation and staining conditions. The
readouts of % positive cells and MFI (minus background) should tell you
which fixation conditions work best for you. Of course, that level of
fixation might not be optimal for all instances where you need to fix
cells - but for each circumstance, you should 'titrate' the fixation
conditions accordingly to determine what works best.

Personally, I find a 'milder' fixation works best to preserve surface
staining of human lymphocytes, but a 'stronger' fixation is needed for
optimal intracellular staining. I don't believe the PI readout you are
using should be the criteria by which you decide on an approach.

Received on Mon May 5 16:18:00 2008