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Some kind of contamination?


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#1 SciCell

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Posted 27 July 2010 - 08:57 AM

Hello all,

I have a silly Q. I have done transduction(infection) to produce a GFP tagged protein in mouse sarcoma cell lines. The pool population thus generated was very good. I did dilution cloning in 150mm plates and 96w plates. Some how I felt the 150mm plates were not healthy after 1 week. I showed it to the scientists and others in our lab who said that it is NOT contamination but there is some kind of protein deposited at the bottom. (Yes it looked like there is some thick coating at the bottom and the cells are embedded in the coating). By the way the cells are in 0.5ug/ml Puromycin selection medium (actually the kill curve for these cells - for the naive ones - showed that a concentration of 0.8 or 0.9ug/ml would kill the cells to good extent. But still we only use 0.5 ug/ml.) However, I threw away the 150mm plates because I was not satisfied. I picked few clones from 96 well plates and transferred them to 24w plates. Last evening, I changed the media in the clones to complete antibiotic -free medium. Now I see the same condition. Should I continue to keep them or throw them away? The clones have beautiful expression of GFP as seen by florescence microscope.

(Anyway I started another culture by thawing a fresh pool population vial and re-did the cloning but I'll save time if I continue with current picked clones.)

Do you have any suggestions? And sorry, I cannot take a picture and post it here.

Thank you.

#2 bob1

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Posted 27 July 2010 - 04:15 PM

It's not dead cells/debris left behind by dying/dead cells? If you still have the plates, can you take a swab and see if it will grow in a cell-free environment (i.e. another plate with the same medium, and perhaps a plate with some fresh unopened before medium).

#3 SciCell

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Posted 28 July 2010 - 09:53 AM

Hi Bob1,

Thank you for your reply. But I have thrown away all the cultures, since I thought I saw some bacteria moving around, though it is doubtful. I say it is doubtful because even though I changed the media into completely antibiotic-free media, the media color did not change after 2 days also, but the cells were over-grown. So I threw away. I am still in dilemma if I did the correct thing.
Is this possible? If the cultures are contaminated with bacteria, - no media color change, and the cells growing to full confluence? I am not sure if I did the correct thing. Anyway, I have new clones growing - so

Thanks

#4 bob1

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Posted 28 July 2010 - 12:38 PM

It was quite likely that the dead and dying cells and even the stuff that you saw moving in the plate was actually cell debris from the dying cells due to selection as puromycin kills cells.

#5 SciCell

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Posted 03 August 2010 - 11:13 AM

It was quite likely that the dead and dying cells and even the stuff that you saw moving in the plate was actually cell debris from the dying cells due to selection as puromycin kills cells.



Hi,

I would like to inform you that those things re-appeared the second time too. This time, I took the supernatant and put them in 6 well plates with complete growth media without any antibiotics - over night. The plate looked clean. Also we did in-house mycoplasma testing which came negative. I reduced the puromycin to 0.25ug/ml that is half of original and still no media color change. If this was bacterial contamination like I suspected, then, I am sure they must increase over night. So perhaps this was like what you said - either cell debris or some protein!!
(But I am still not satisfied. Protein or cell debris has no motility. Right? I saw something moving!!)
Well whatever, the problem seems to be solved.

Thanks

#6 laurequillo

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Posted 03 August 2010 - 12:23 PM


It was quite likely that the dead and dying cells and even the stuff that you saw moving in the plate was actually cell debris from the dying cells due to selection as puromycin kills cells.



Hi,

I would like to inform you that those things re-appeared the second time too. This time, I took the supernatant and put them in 6 well plates with complete growth media without any antibiotics - over night. The plate looked clean. Also we did in-house mycoplasma testing which came negative. I reduced the puromycin to 0.25ug/ml that is half of original and still no media color change. If this was bacterial contamination like I suspected, then, I am sure they must increase over night. So perhaps this was like what you said - either cell debris or some protein!!
(But I am still not satisfied. Protein or cell debris has no motility. Right? I saw something moving!!)
Well whatever, the problem seems to be solved.

Thanks


Just for curiosity, did you notice any funny smell? Because I am having a similar problem, but I am quite sure mine is a contamination because of the smell, but I have no idea where it came from ( I checked every step).
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#7 bob1

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Posted 03 August 2010 - 02:13 PM

Debris can move by Brownian motion which is the temperature dependent motion of particles in suspension caused by interactions with molecules moving around in the fluid.

#8 SciCell

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Posted 03 August 2010 - 04:08 PM

Debris can move by Brownian motion which is the temperature dependent motion of particles in suspension caused by interactions with molecules moving around in the fluid.


Wow! This is some information for me. I didn't know debris could have movement. Yes, the movement was like brownian movement - so I thought bacteria. Now I am scared to use high concentration of Puromycin.

laurequillo:

No. There was no smell at all. Everything looked normal except for these moving things in culture!

Thank you all.




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