Hello everyone,
I ask for your guidance regarding these unknown black dots that seem to be in my cell flask.
The cells are macrophages derived from human blood, and the culture is around 2 weeks old. They black dots have been seen from start (as in seen in the flask directly after isolation) and seemed to be adhered to the cell flask even after multiple washes with PBS. They do not move and the cell media (containing pen/strap) is clear and not yellow.
Any idea on what it could be?
Thank you for taking the time reading this.
Best regards,
Elina
What is your method of isolation, are you sorting monocytes or just doing the plastic adherence method?
Could be from platelet contamination, and the platelets have then been activated by FBS stick and clump to flask. Could be other adhered cells that have are now dead and have their contents have burst stuck to flask. Bacteria/Fungi would usually float, and the size seems a bit off for them?
@Charlie Bridgewood just doing the plastic adherence method!
I suspect platelet contamination as well, but unsure on how it looks like in a culture flask. I agree that that the size seem off for it to be bacteria or fungi. It seems like the black dots are behind the macrophages, not sure if that changes anything.
Yes, the platelets will stick to everything, including your monocytes. The type of tube you use to collect blood effects platelet activation, best to use EDTA and not other types such as Heparin. After you isolate PBMCs, it is good to do a slow spin (200g) as this means the pbmcs pellet and the platelets do not or can lyse them with a lysis buffer that you use for red cell lysis.
Charlie Bridgewood Thank you so much for your help and input! I will definitely try both using EDTA and a slow spin to see if the problems remains. Cheers!
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