Dear all,
I am facing a particular problem while preparing the cell line MDA-MB231 for confocal microscopy. I use 3% paraformaldehyde for fixation. I wash them with serum free medium before fixing them to make sure there is no morphology change. I observed my cells after the fixation step and some of the cells looked bright and I am failing to interpret this. Can this mean that my cells were already dead before I fixed them or did their morphology changed during the fixation step. I am attaching an image for reference. Please take a look. Thank you very much in advance.
Hi Udesh,
Looks like your cells have detached from the surface. Make sure you pipet and aspirate very carefully when removing the media, washing and adding the PFA (if this was the problem though, you should always have the problem when making any manipulations).
You can try prefixing the cells by simply adding some PFA (25% of your media volume) into the well and incubate for 2 min. Then aspirate, wash, add PFA to the cells and incubate for 15 min. Try using freshly prepared 4 % PFA maybe.
Cheers, Laura
Dear Laura, Thank you for the answer. However, if the cells have detached, they should be removed when I washed the cells with PBS after the fixation step. Contrary, they remained there like they are adhered on the substrate. Nevertheless, I will give your protocol a try. Thanks a lot !
Well, yes, they still have some connection to surface or neighboring cells. In this shape, it won't be very strong though ;) Solve your PFA in PBS and adjust the pH to 7.4.
In my experience, MDAMB231 cells do not attach well and are easily disturbed..
In your case, I think they still adhere to the surface, but barely ; they are round instead of being flat.
I have the same problem...
Maybe try to pipet gently on the side of the well ?
Thanks Laura, I'll be more careful next time.
Dear Mr. Benjamin, I have been working on this cell line for several months and in my case they have adhered firmly on to the surface each time. Some time when I subculture them, I have to tripsinize them twice and tap the culture dish hard to make them detatch from the surface of the culture dish.
The cell line's normal morphology is fibroblast-like. I think it is fair to assume that they detached from the surface which I was washing them and hence they look flat. Nevertheless, I'll try being more careful next time and see if the problem persists. Thanks All ! Cheers!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line