Hello everyone,
I am trying to do immunocytochemistry on PMA differentiated U-937 cells, a monocytic cell line.
After fixation, I can see shadow-like bubbles next to each cell that disappear after permeabilization.
What I am concerned is that if the bubbles are leaking cytoplasm, it might mean that the cellular structure is destroyed.
Some fixed cells look collapsed after the fixation.
Does anyone have similar experience? Is there a way to mitigate the issue?
Here are my fixation methods;
Cells are washed twice with PBS containing Ca2+ and Mg2+.
Fixed with 3.7% paraformaldehyde in PBS containing Ca2+ and Mg2+ pre-warmed in 37C bath, fix for 5 minutes.
Washed twice with ice-cold PBS containing Ca2+ and Mg2+.
Wash with ice-cold PBS containing Ca2+ and Mg2+ and incubate for 5 minutes. Wash two more times.
Thank you.
Hello,
in my opinion it looks like debris. So I would think some cells are dead. We use two different methods for fixation of cell culture and tissue.
1. method:
- wash with PBS 3x 5 min (cell culture) up to 3x 10 min (tissue)
- fix with ethanol/acetone (1: 1; or methanol/acetione) 90 sec up to 30 min
- wash with PBS 3x 5min up to 3x 10 min
--> all components are 4°C pre-cooled
2. method:
- some antibodies only work with PFA
- wash with PBS 3x 5 min (cell culture) up to 3x 10 min (tissue)
- fix with 4% PFA (in PBS without Mg2+ and without Ca2+
- wash with PBS 3x 5 min (cell culture) up to 3x 10 min (tissue)
You can also use instead of PBS 0,1 M phosphate buffer...
All components are 4 °C pre-cooled! Do you checked your 4% PFA solution for pH? It should be 7.3?!
best regards,
constanze
Hi there Wonyong Lee
U-937 are cells in suspension. Are you fixing them in suspension or are you immobilizing them on coated surfaces prior to fixation? The PMA differentiated macrophage like cells are known to give out extracellular vesicles, and those are maybe what you see as small shadow like blobs.
Handling stress may exacerbate how much extracellular vesicles these cells generate.
Now it depends what you want to image in these fixed cells. Of course the extracellular vesicles will carry some cytoplasm of the parent cell, but that need not be an issue, if what you expect to see is not super low expression.
I think your fixation protocol is alright but certainly try what Constanze Nossol suggested to see it if helps.
Regarding leakage of cells, to quickly check that incubate your cells with a live/dead stain or trypan blue which enter cells with ruptured/damaged membranes only. Cells with intact membranes remain negative for uptake of these dyes. Normally, macrophages giving out extracellular vesicles do not have rupture in their plasma membrane and hence are not positive for trypan blue or any other live dead stain.
For the other macrophage cell lines we use, I have seen such a profile as well and they are fairly sensitive to handling. Avoid mixing reagents at different temperatures- either all at RT or all at 4 degrees Celsius.
Be ultra gentle with centrifugation and agitation during washes (pipette gently).
If you plan to study surface proteins or some lipid based molecules, avoid using any organic solvents for fixation, as they extract the lipids and hence some proteins which have GPI anchors or associate with lipids may be lost. You will also lose actin staining with fixation using organic solvents (methanol/acetone)
Hope this helps
Best
Aparajita
Seems that it is not bubble. Sort out the target cells by centrifugation and transfer in vial containing desired fixative. shake the vial for homogeneous suspension and then load the slide. Spread it so that the almost mono layer is formed and air dry. Hope it will solve your problem.
Are you mounting the cover slip immediately after washing? If so then it is possible that these are bubbles. Try drying for a few minutes before mounting. Another possible reason could be use of excessive mounting solution.
Hello Sir,
I think you should try using PBS without Ca and Mg. In my case when I was working with isolated hepatocytes for ihc we always used PBS w/o Ca n Mg. If we used PBS w Ca and Mg then the cells would collapse. My experience is use PBS w/o Ca and Mg.
do you get this stuff on regular basis or just once? the photo is bit confusing. Can you draw a cell with pencil on a paper and label exactly what bubble means and where is cell in relation to bubble, thanks
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