If you are doing fixation at room temperature, then do it for minimum 1 hr instead of 10 mins. All the solution like PBS or 4% PFA should be at 37 C. The standard timing for fixation is 1hr at 37C or overnight at 4C, in the case of 4% PFA.

For cultured cells, you should use PBS with Ca++ and Mg++ ions.

Because if you are washing grown cells with PBS without Ca++ & Mg++, it will cause the rounding and detachment of the cells from surface.

 Hope this will help you.

Regards

Dear Monika, the cell wasn't rounded/detached after first wash with PBS. But it became rounded/detached after the fixation and before wash it again with cold PBS.

I do normally wash with PBS in this type of cell before, in culture plate, and it won't be detached at all.

I use 4% buffered formaldehyde to fix my cells, and rarely have problems. I fix for 10 min at RT, then wash 2x with PBS. Never fix longer that 10-15 minutes, because the formaldehyde can affect the staining of the actin cytoskeleton. I do not see changes in cell morphology before/after fixation, so it should work.

By washing with PBS you do increase the chance of washing away cells; this depends on your cell line. It may still affect your cells after you have checked with the microscope.

best of luck

I had this problem before. I changed how I fixed the cells. First I removed half of medium (500ul) and I add 500ul of pfa 4% for 5 min. It means I pre-fixed the cells with pfa 2% (because pfa 4% was diluted in the medium). Then I removed all the medium and I add 300ul of pfa 4% for 15min. After that I washed the cells with pbs 3 times. It worked for me.

Hi Asa,

I agree with Michiel, it is unlike that the short time of fixation is the problem. I never fixed for longer than 15-30 min and I never add this kind of problem. And if the morphological changes happens in 10 minutes PFA, fixing for longer time is not going to help you, it will just "fix" stronger your rounded cells. 

I cannot be sure but I suspect that your PFA solution is gone (or at least not 4% anymore). It would be a good idea to try a fresh prepared solution and as suggested by many people, if the cells are kept at 37ºC before fixation, try to keep all the solutions at 37 ºC to reduce the stress. Another important point to check is the osmolarity of your PFA, if it is ipertonic it would cause cell shrinkage and if it happens quicker than the fixation you have the problem you have.

Finally, if all this does not work, it could be that your cells are to delicate and that you need a faster fixation method. You can use glutaraldehyde that should work in seconds however this fixation does often interfere with epitope recognition from antibodies.

Hope this help.

Good luck!

Hi Asa,

I also suggest 10-15 min fixation with fresh, warm PFA solution. 

If this doesn't help you could try fixing with ice cold methanol for few minutes at -20C. That can change your epitope recognition so might not work with all antibodies.

Good luck!

Asa, 

try the following

1) Make sure that your PFA solution is always fresh. It should be prepared from the powder and used or discarded within one week at most. 

2) PFA causes cell shrinkage and subsequent changes in morphology. To compensate for this shrinkage try to prepare PFA in a hypotonic solution. I generally use 0.5x  to 0.75 x PBS when making 4%PFA  

Hope this helps

Asa,

I suspect the you are already doing this correctly, but since you did not specify in your description I should mention it here. Be sure that your 4%PFA was dissolved in PBS. If you are working with a 4% PFA that is only in water it can cause detachment. It is an easy error to make, and in my experience this causes a pretty dramatic detachment.

Otherwise it could have to do with your 4%PFA being old (ie not doing a good job of fixing) as mentioned above, although I have not personally had a problem with that.

As far as fixation time, I agree with some above that it is not likely the issue. formaldehyde penetrates fast, and more often I have run into issues with over-fixing cells or tissue. Over-fixing results in things like poor antibody penetration, antigen degradation (denaturation?), and weakened/loss of GFP fluorescence (if cells were expressing GFP, etc) . In the past, I have typically used a 10 minute 4%PFA in 1XPBS fixation for culture cells with success.

in addition to making sure you use PBS, you may want to try making the PFA using NaOH to raise the pH and no heat (as if you accidentally get to hot, it will break down the PFA). Add your PFA powder to PBS on a stirrer, add a few drops of 2N NaOH to bring the pH up. It should go into solution pretty quickly. Then top up with PBS and use HCL to bring pH back to 7.4. We filter out any remaining particulates and freeze with no issues. 

Hello Asa, I like to say few changes in your protocol.

1. Precoating the cover slip for 0.2% gelatin.

2. Fresh 4% PFA, you can store +4C for 1 week.

3. Fixed the cell for 10 minutes on ice.

Hope this will help you. Gud luck.

Hi Asa, 

I agree with many of the answers above, I am using a sandblasted polystyrene for my experiment and it affects cells attachment negatively so I have to take extra care when washing the cells or changing the mediums. 

The following way helped my greatly, When I remove any solution, I remove only half  of the amount, meaning that if it was 1000 ml I only remove 500 and add the next required solution and so on. This helps in keeping the cells wet and you avoid the damage that can occur by removing the solutions and completely drying the samples (which can be a cause for the damaged cells). 

I hope that helped. 

Thank you you all.

4%PFA was diluted in PBS. I will try the experiment again in a few day and let you know what is the cause.

Hi Asa,

Besides PFA, I think it may have to do with what your coverslips are coated with. I am currently working with a BE(2)-M17 cell line (also a neuroblastoma cell line). Cells adhere nicely to plastic plates but not glass coverslips. Similar to what you describe, as soon as I added PFA, they've all detached.

Reading through some of the papers that report using this cell line, they recommend coating coverslips with Fibronectin or Poly-Lysine D. I haven't tried it yet but I think that should solve the problem. Coating is typically done a few days or a week prior to seeding cells, so it's a fresh batch each time you want to use them.

Hope this helps, Good luck!

Hi Asa,

Try to fix cells with iced-cold PFA (fresh prepared or store at 4ºC for about 1 week) and not leave it at RT as you did; also extend fixation time for 20 min.

Good luck!

Adriana

Dear Adriana, It is extremely important to use fresh solutions, namely, prepared in  the same day. Additionally, for cell culture I recommend the  paraformaldehyde of EMS (Electron Transmission Sciences) in vial.  The concentration used in my lab is 2% for 20 minutes at room temperature.

Hello Asa,

Your problem is not complex and I really appreciate the suggestions already laid by people above..The solution is really simple. But first let me address some of points discussed above...You have no need to do any coating since cells were growing good and everybody knows out thr that cells this cell line grows good without any attachment factor. No need to make PFA Fresh everytime as making PFA is also a time taking process, we can use stored PFA and we have used it multiple times successfully. No need to try long time incubations (but it's good if you do longer incubations as it will fix cells well, but if ur experiment don't allow u to wait for long..15 mints is just fine)...and last but not least, thr is no need to supplement PBS with Mg or ca and they can have some unwanted effects on cell staining specially when ca is concerned.

Your problem is just because you used cold PFA...Whenever we add cold PBS or media to cells you can observe rounding of cells. If cells will contact with anything cold they will shrink and become rounded thats why some of ur cells lose morphology and even retracted back neurites..When cells are alive, anything cold will result in cell death and cell stress. So at RT 15min-1hr fixation in 4% PFA (37 or room temp-not COLD) is suffiicient to achieve fixation...if you want to do fixation at 4 degree then put the PFA in cells and after some time put the cells in freeze. I guarantee you that they won't dettach/lose morphology then.

Hope it helps !

Dear ASA, you've got very nice tips abov. I would add that you may try to fix the cells with fresh warm (37 C) PFA at room temperature as described above but avoiding the PBS washing step. I work with actin ultrastructures and noticed very often that cell morphology is better maintained when the first washing step is not performed. Slight changes in the PBS osmolality can lead to changes in membrane/subcortical actin resulting in altered morphology.

Good luck.

Hi Asa,

You should be confused right now....

I just want to add some notes to this discussion that I think it could be relevant: sometimes we need to fix cells in ice using iced-cold solutions in order to stop all metabolic processes. This is crucial for some studies ...namely cell signalling studies. The key is to fix cells immediately after removing them from culture (eventually a PBS wash should be done but it takes no more than 1min). I do not work with your cells, but I worked with several other cell lines and primary cultures seeded on plastic or glass and I always do the same:

1. Remove cells from culture and place on ice

2. Remove medium immediately and wash 1min with iced-cold PBS

3. Fix with iced-cold 4%PFA (diluted in PBS, do not need to be freshly prepared) for 20 min. (5 min after adding PFA I remove cells from ice and let the last 15 min at RT)

For me, it always worked...but, once again, I do not know your cells. I think you should try all the solutions that have been presented here and choose the one that your cells like more.

Good luck!

Adriana