I have performed the primary cell cultures from wild-type Danio rerio blastomeres using DMEM (medium). The results of cultures observed with inverted microscopy showed not very good results. Here are some related questions about it.
1. What might cause the cell is not attached (adherent)? Is contamination capable of causing non adherent cells?
2. What do you think about the images I have included? Can the contamination be determined from bacteria or certain organism?
3. What are the obstacles to cells to reproduce/proliferate themselves?
4. What are the factors influence the cell signaling and interfere division and differentiation?
5. Is it bad if the incubation was done in 28 degrees of Celcius?
Hi,
The bacteria and fungi contamination is not present, but the cells are dead.
Komórki doesn't attach to the plate because they are dead. Try to choose the cells culture conditions better.
1) Try to use:
a) Complete Growth Medium contain: 50% Leibovitz's L-15 medium (ATCC 30-2008) 35% Dulbecco's Modified Eagle's medium, high glucose (GIBCO 12100) 15% F12 medium (GIBCO 21700) The above are all without sodium bicarbonate Supplemented with: 0.18 g/L sodium bicarbonate 15 mM HEPES 10% heat-inactivated fetal bovine serum
b) Freeze medium contain: Complete growth medium, 85%; additional HI-FBS, 10%, DMSO, 5%
c) Passaging with Trypsin-EDTA solution.
c) The cells should be cultivated at 28°C.
Good luck!
Hello Pratama,
This paper may be helpful for you!
Embryonic zebrafish primary cell culture for transfection and live cellular and subcellular imaging. Dev Biol. 2017 Oct 1;430(1):18-31. doi: 10.1016/j.ydbio.2017.07.014. Epub 2017 Aug 9.
Cheers,
Philip
Hi,
The bacteria and fungi contamination is not present, but the cells are dead.
Komórki doesn't attach to the plate because they are dead. Try to choose the cells culture conditions better.
1) Try to use:
a) Complete Growth Medium contain: 50% Leibovitz's L-15 medium (ATCC 30-2008) 35% Dulbecco's Modified Eagle's medium, high glucose (GIBCO 12100) 15% F12 medium (GIBCO 21700) The above are all without sodium bicarbonate Supplemented with: 0.18 g/L sodium bicarbonate 15 mM HEPES 10% heat-inactivated fetal bovine serum
b) Freeze medium contain: Complete growth medium, 85%; additional HI-FBS, 10%, DMSO, 5%
c) Passaging with Trypsin-EDTA solution.
c) The cells should be cultivated at 28°C.
Good luck!
Im agree with Justyna.
1) Use trypan blue to verify viability of ur cells (if they are death, not attached its consecuently)
2) The media DMEM alone not are recomended:::.. try to use:
50 % L-15 + 35 % DMEM HG + 15 % Ham's F12. All without sodium bicarbonate Supplemented with: 0.15 g/L sodium bicarbonate, 15 mM HEPES ,0.01mg/ml bovine insulin, 50 ng/ml mouse EGF, 5% heat-inactivated fetal bovine serum, 0.5% Trout Serum
Do not filter complete medium.
Or another more simple like this: DMEM:F12 (50/50) + 10% FBS
Or DMEM + 15% FBS.
3) to detach this kind of cells some times EDTA coud be malicious. (but this is a primary culture that u need to evaluate those things). hehehe!!!
4) incubation its great at 28 (can be variant between 26 to 29).
I hope it helps!!!
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