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THP-1 differentiation using PMA yielding black dots within cell structure - affe

black dots in cells black particles in cell thp-1 macrophages contamination?

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#1 daywalkerbyday

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Posted 02 July 2013 - 06:07 AM

Hi all,

I've been using THP-1 cells for the past 3 years and up until recently have had little to no trouble differentiating them. However, 6 weeks ago, I noticed that black dots are now appearing within the differentiated cells. These particles are static, are not causing apoptosis, but results generated since the appearance of these particles don't correlate with previous work - i.e. qRT-PCR data that has remained unchanged. I've changed media, FCS, broke out new cells, all to no avail.

I have attached a 'before and after' picture. I am going out of my mind. If anyone can shed light on this I would be extremely grateful.

Thanks!

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#2 bob1

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Posted 02 July 2013 - 12:59 PM

They look like vacuoles of some sort, but the photos are quite unclear. Perhaps if you attach the original photos it will be easier to see.

#3 daywalkerbyday

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Posted 03 July 2013 - 03:23 AM

Thank you for responding - I have gotten vacuole formation along with the appearance of these black dots, but whether they are before or after I can't be sure. Caspase 3/7 assays and trypan blue are both normal. I've uploaded the original file - this was a co-culture experiment which is why you see a different cell type alongside the differentiated macrophages. One slide contains 'normal' THP-1 macrophages while the other, the black dots are apparent. There is quite a bit of background however this was the microscope itself.

Thanks you so much - I feel like I've been banging my head off a brick wall.

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#4 bob1

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Posted 03 July 2013 - 12:31 PM

Having other cells in there would swamp out the macrophage signal in a caspase assay, which may be why you aren't getting any result from that. The cells do look very unhappy and I think they are probably either senescent or could be apoptotic, with some condensation of the cellular components (possibly ER) into the black dots that you are seeing.

#5 daywalkerbyday

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Posted 03 July 2013 - 01:47 PM

The caspase assay was performed on THP1 macrophage cells alone, as was the frypan blue assay. Senescence is one possibility as DMSO caused activation of caspase 3/7 with little to no cell death on trypan blue.

Unfortunately, these are the only pictures I have to illustrate this 'phenomenon' so to speak. The dots tend to cluster around the nucleus and along the processes in a defined manner. I can't think of anything they could possibly be that I could stain for/measure to figure it out.

I'm also getting particle formation usually between 24-48 hours post PMA treatment. I've sourced PMA from a second lab and that's produced the same results. As a last port of call, new PMA has been ordered to out rule DMSO contamination in 2 independent labs.

Cells seeded earlier today with a more dilute concentration of PMA contained some vacuoles that appeared to be dancing, suggesting an intracellular contaminant which I've never seen. This was witnessed on one batch of cells alone, with 3 further batches tested.





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