I'm new to cell culture and i have started with Lc540 leydig cells i started growing the cells and i notice these black dots as shown in the picture in red arrows and it doesn't affect my culture and i'm sure its not contamination too. The other yellow line indicates hell a lot of bubbles inside the cell and if i even split the cells its healthy... Please do advice me on what these are and how can i get rid of these ......
If your cells look completely different than they did in the past, then they may be stressed by the culture conditions. You should make sure you are using the right media and that you are splitting them correctly; make sure they don't get overgrown, but also don't plate them too sparsely. There is also a possibility that the cells have mutated or you have contamination with a different cell line. In this case, you will need to start a fresh culture. That being said, while I've never grown LC540 cells, the image you show doesn't look too abnormal for an adherent cell line. The "bubbles" are likely a variety of endosomes or other subcellular structures. I don't know exactly what to call the black dots, but they just look like normal material that cells generate when they are proliferating. If the cells are growing well and work in your experiments as expected, I don't think you have a concern.
I haven't worked with the specific cell line, but there are cells that bleb and macro- and micropinocytose a lot, and being the LC-540 transformed cells this is very well possible and likely. I can't really make out the cell shape, there is not enough contrast, anyway, what looks like with your cells is that they are "drinking" a lot. Bubbles are normally macropinocytosed medium. The smaller blackish spots are generally smaller vesicles derived from micropinocytosis. In particular macropinocytosis is Ras-stimulated represents an important route of nutrient uptake in tumours, it's a fast feeding route!
http://www.nature.com/nature/journal/v497/n7451/full/nature12138.html
Thanks Pietro Pilo Boyl for your answer. I too had the same thought these particles are due to process of exocytosis as i read through it vague. But, then i couldn,t get clearly what do you mean by cell drinking.
Thanks Micheal.... hope you made me clear, i shall start off with my experiments.
:-D
We say that cells "drink" a lot when they have this massive macropinocytosis that produces the large bubbles you observed... just a way of putting it in common words...
Good luck with your experiments
Hi, The black spots may be Mycoplasma contamination. The bubbles, Leydig cells have some special crystals called as Reinke crystals, also store cholesterol for steroidogenesis. Think in these aspect. All the best
Anbalagan Moorthy
Thank you so much for your comments.. indeed much helpful (Reinke crystals)
Thanks Pietro... I'm new to cell cultre and so was difficult to get ur common terms :) :)
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