Dear Sebastian,

Please read the following text:

https://www.quora.com/What-kinds-of-structural-damage-can-one-cause-to-cells-by-preserving-them-in-formaldehyde

In the simplest sense, formaldehyde or formalin reacts with the various functional groups of biological macromolecules in a cross-linking fashion.

 This is a two step reaction:

The reactive sites of biological molecules form mono, dimethylol or hydroxyl methyl derivatives. The subsequent cross-linking occurs by the formation of a -CH2- methylene bridge. Formaldehyde can react with the following types of reactive groups:

Primary amines

Amides

Alcoholic hydroxyl groups

Sulfhydro groups

Aromatic rings

There are various protocols available for the chemical fixation of biological samples by formaldehyde, and each of these protocols may exhibit different kinds of structural anomalies in the cell. The correct protocol must be selected carefully to avoid artefacts in cell morphology and molecular structures. Examples of some of these artefacts are below:

Effects on cell morphology:

Cell or tissue shrinkage is a common occurrence in aldehyde-fixed samples, and causes alterations in the structure and localization of the organelles, and various cytoskeleton- and membrane-associated structures. The best way to correct for these artefacts is to optimize the fixation protocol depending on the end goal of each study.

Vesiculation or "blebbing" of cell membranes can be seen in aldehyde-fixed cells, and may be a consequence of a local breakdown of membrane-cytoskeleton coupling due to lower PIP2 levels in the plasma membrane.

Fixation with paraformaldehyde (left) induced large blebs, whereas fixation with KMnO4 (right) did not induce bleb formation. Image source: Fixation-induced cell blebbing on spread cells inversely correlates with phosphatidylinositol 4,5-bisphosphate level in the plasma membrane

Deformation of the cytoskeleton may be caused by aldehyde fixation. In particular, microtubules appeared to be "curved" or bent in aldehyde-fixed cells when compared to live cells. This suggests that subcellular positioning of various organelles and also membrane topology may be affected in aldehyde-fixed cells.

Effects on cellular chemical composition:

Leaching is a common phenomenon in cell/tissue fixation, and refers to the loss of small molecules from the cell due to membrane permeabilization. The total effects of leaching vary with the chemical composition of tissues. Molecules that are commonly "leached" include proteins, amino acids, phosphates, carbohydrates, inorganic ions, ATP, fatty acids, unsaturated lipids or lipid oxidation products, etc.

However, out of various fixatives, formaldehyde has been found to best preserve cellular integrity in comparison to a live cell.

Effects on nucleic acids:

Several studies have found that formaldehyde fixation, if done correctly, does not cause complete degradation of DNA or RNA, although the formation of cross-links make the fixed samples unsuitable for detection by DNA or RNA probes. I've discussed this in more detail here.

Effects on protein structure:

Protein secondary structure appears to be "locked" in place by formaldehyde fixation. This was demonstrated by the absence of significant changes in the infrared spectra of fixed proteins.

Tertiary or quarternary structures are also "locked" by the formation of cross-links in globular proteins, although this usually results in inactivation of the protein's functions.

Sources and further reading:

Chemical and physical basics of routine formaldehyde fixation

Chemical alterations to murine brain tissue induced by formaling fixation: implications for biospectroscopic imaging and mapping studies of disease pathogenesis

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy

Reversible and irreversible effects of chemical fixation on the NMR properties of single cells

Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation.

Formaldehyde fixation

Direct comparison of common fixation methods of microtubules in zebrafish embryos

Fixation-induced cell blebbing on spread cells inversely correlates with phosphatidylinositol 4,5-bisphosphate level in the plasma membrane

Aldehyde fixation causes membrane vesiculation during platelet exocytosis: a freeze substitution study

Fixation-induced redistribution of hyperphosphorylated RNA polymerase II in the nucleus of human cells

Kinky microtubules: bending and breaking induced by fixation in vitro with glutaraldehyde and formaldehyde

Immunolabelling artifacts and the need for live-cell imaging

Hypothesis for the influence of fixatives on the chromatin patterns of interphase nuclei, based on shrinkage and retraction of nuclear and perinuclear structures.

Hoping this will be helpful,

Rafik

Hi Sebastian,

first: It's relativley important to either prepare your PFA freshly or make a Formalin like formula (meaning stabilized with Methanol) and then dilute to your appropiate concentration. Formaldehyde tends to react with itself after a while and forms the polymer again, especially at colder temperatures (so don't keep it in the fridge). After a while you don't have any reactive compound left. Nontheless I have never seen the cells beeing destroyed by this.

If your main problem is detachment try warming all your buffers before putting them on the cells - some cells (e.g. Hek293) really don't like it cold and just quit playing once the get cold.

Also just to be sure try pipetting extra carefully to not introduce too much turbulence.

Ok that's it from me. I hope this helps somehow. Let me know how it goes

Best

Björn

P.S. Oh yeah, you can also try other fixation methods (e.g. Ethanol, Glutaralaldehyde etc.) ;)

It sounds your fixative does not work.It seems to me you mix something. PFA is a power, you should measure it and dissolve in PBS. You probably did it i just did not see. pH is low a bit, should be 7.2-7.4 that can be another cause besides cold. And yes in parallel try cold methanol/ethanol fixation. 

Hello Sebastian,

one suggestion from my side: If you have trouble loosing cells during washing steps, try adding 1mm Calcium and Magnesium to the PBS (see here: https://www.researchgate.net/post/Does_PBS_buffer_have_any_effect_on_cell_adhesion). So you can prevent damage to your cells and loosing them even before fixation. 

Best 

Jan

Hi Sebastian,

I am seeing this too as I fix dissociated mouse embryo cells (E11.5). After 10 min in 4% PFA at RT, there is a ton of debris. Do you ensure that everything is room temperature before you start? Perhaps this has something to do with it. I made PFA fresh and still see the issue.

Lindsey

I get similar effects with 4%PFA:PBS in mouse embryonic stem cells. It's not a case of them being "washed off", as others have suggested. I watched my cells under the microscope after adding PFA, and they detach over the space of 2-3 minutes. I will try adding calcium as Jan suggests, but there is also a problem with damage to the cells as reported by the OP. My thoughts are that this may be caused by poor fixation of the cells, in combination with the high osmolarity of the PFA solution. Will post again if I can find the cause of the problem.