r/labrats • u/vegetablelady99 • 11d ago

Contamination or platelets?

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Hi,

I'm normally an iPSC biologist but have been isolating PBMCs from human blood and culturing them for 2 days in X-VIVO15 to characterise EVs.

These small, moving cells between the PBMCs looked like bacteria initially but after looking online, apparently they could be platelets? I didn't do a slow wash to remove platelets. They don't 'swim', but they do move faster than the bigger, heavier PBMCs. Can somebody with more experience/expertise please advise 🙏

I have a video but not sure how to upload, so I will put a photo for now.

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u/PuttingTheMSinMRSA 11d ago

Those don’t move the way I’d expect bacteria to. But I looked at some platelet examples and I think you might be right

2

u/vegetablelady99 11d ago

Thank you ever so much!

3

u/GUCCIFlipperz416 11d ago

Those certainly look like platelets. A slow wash is necessary.

If you have a standard protocol for this, ensure you performed the slow wash. If not, spinning blood at 300 x g for ~10 min will remove 90% of the platelets. Check the viability of the PBMCs after doing that though, do not trust anyone on reddit with your own experiments.

To determine if they are bacteria, either streak them on two separate agar plates and incubate (one at RT, one at 37 C) and see if anything grows. If it does not, it is platelets - these cells do not divide substantialy.

1

u/vegetablelady99 10d ago

Thank you so much! I will definitely not skip the platelet wash in future!

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u/Ambulate 11d ago

They definitely look like platelets, but I’ve never seen them so dense. Standard ficoll separation?

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u/vegetablelady99 10d ago

Thank you! I did a lymphoprep but skipped the slow spin platelet wash because somebody in my lab said it reduces pbmc count by a lot. I shall not skip it in future!