This is the second time in 6 months my cells have formed a black/brown aggregate that grows with time. Details:
Neurobasal A (with B27) and gentamycin, FuDR added at DIV4
Neurons DIV4 taken from P1 C57/B6 cortex (includes hippocampus)
1mg/ml poly-d-lysine coated (and washed) prior to plating 384 well Greiner uClear plate
20m digest with papain followed by 4 washes and 4 triturations with P1000 tip (done this part 100s of times with no problem). Cells filtered through a 40um filter before being spun down (at 420g) and counted. Plated at 12,000 live cells per well (384 well plate)
Cells were transduced DIV0 with AAV9-CamKII_Cre (frequently perform this transduction without these symptoms)
Some wells are perfect with no aggregates. But most contain at least some aggregation. Other wells are completely aggregated. No spike in phenol red color, cells look healthy in the well but the aggregate will enlarge over them. The small aggregate pieces move and jiggle. The large pieces of aggregate are stationary.
Anyone know what this is? Contamination? Stress granules? Mold? Debris? Microglia activation?
Anything would be appreciated...
Hi, if it is heavily pigmented (black/brown), it is quite possible, that it may be a fungal contamination (eg. Aspergillus niger). Spores are ubiquitous in (lab) air. Did you observe fungal filaments. If it is a mold you may add amphotericine to prevent mold growth, but this agent cab dramatically interfere with neuronal functions and biochemistry.
Good luck!
Thanks for your help Just Genius. I have never observed any filaments, just globular bits that grow asymmetrically. I looked up Aspergillus niger, does fungal contamination always grow spherical? I've never seen large rounded objects (such as in the examples of Aspergillis niger online) only raggedly shaped objects.
It is very heavily pigmented though...
95% these are just cells that failed to attach to the surface. Usually neurons tend to clump together in these brown-ish aggregates when the polylysine surface is not good, or degraded (do not use if prepared more than 2 weeks before) or coating was insufficient (use fresh poly-K or gelatin + polyK). At some point the cells dye and stay there as floating clumps (that's why the color is dark). I have seen this happening in my cultures from time to time....
Hi Kyle -
I have seen this as well when deriving neurons from stem cells. It is possible that the aggregates are embryoid bodies - not typical and smooth and potentially layered with brown fat. The fat accumulation could be due to a number of factors in your protocol, but it is a potential energy source for the cells. Do the layers persist for more than a couple of days/shrink or grow?
You can collect a sample and run a few tests for stem cell markers to determine if they are in fact embryoid bodies and which germ layer they are expressing.
hello every one
I have this problem in my cell culture and evryday can be increase ! this problem comes other lab to our lab with hela cell and I don't know what is this?
Hi,
I had similar problems when I used PDL for my neuron cultures. Inconsistent culture quality and huge variations in adherence. I agree it's probably because your PLL is too old.
My lab switched to coating our plates with dPGA from Dendrotek (https://dendrotek.ca/products/centrifuge-tube) and it solved a lot of our inconsistency problems. Cells adhere more consistently and our cultures last way longer (10-11 weeks for dPGA vs. 2-3 weeks for PDL. Also, I can keep a coated plate with water in it in the fridge for months with dPGA and it still works just as well when I want to add cells as an overnight coating would work. Now I just do all my coating for the next 4 months at once and keep the plates in the fridge until I need to use them.
Hope this helps!
so does it mean that they are not infection, cause you experience to see less of them after using dPGA ??
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