I have been growing neuroblastoma B50 cells on glass coverslips and the cells appear healthy when I see them under the normal microscope. For electrophysiology, I take the coverslip out and place it on the stage of the recording microscope, and dip it with the same culture medium that it was inside the incubator. Then I see the cells under a 40X objective. To start with the cells look okay, and then suddenly their membranes start to rupture. At the end of 5 mins, they look like they have burst (I am attaching an image of a burst cell that I took with my mobile from the computer screen). If I wait longer, all of them burst completely. This effect is faster if I dip the coverslip in fresh DMEM (in this case they burst within 1 min).
Few other details: Osmolarity of the growth medium is 320mOsmol. Another thing is the temperature of the recording stage is 27deg.
Can the drastic temperature difference be the cause? Or the pH of the medium changes so suddenly after taking it out from the incubator? Can someone please help me in this regard?
Thanks heaps!
What do you mean dip the slide in medium? Only a thin layer of medium remains afterwards?
I do not think cells burst due to pH changes (in any case, you can use medium with HEPES buffer to avoid pH changes, they can affect vehicle transport etc). Temperature neither.
If you only have a thin layer of medium, it is very easy for osmorality to change drastically due to evaporation. That would not make them burst (grow in volume until they explode) but the final effect could be like the one in your image. Another point would be if during manipulation they are exposed for too long to air before you ut them back to medium. The effect of this kind of death is not aways immediate.
Cheers
Hi, I thank that cause the burst cells duo to variation in temperature between medium and incubator and the cells haven't ringer solution around it.
Ensure that your cover slips remain moist.
Avoid direct exposure to air.
- Badges
- Science method