Contamination we can't see...??? - (Feb/24/2011 )
We routinely extract astrocytes from brain and this protocol has worked for years!!! Recently, a problem started occurring intermittently (we get a bad prep and then a good prep and then a bad one...and so on). When we plate the cells (P0), they look amazing!! We let them grow and they appear really happy. When we split them, they look happy for a couple of days and then...they come off!! The few cells which remain attached look granular and weird....The media doesnt turn turbid even after weeks in culture but the surviving cells just don't divide anymore. We don't routinely test for mycoplasma or viruses, but if it was contamination, why would it only affect the P1 while the P0 are having the time of their lives? Or is it some weird contamination which doesnt affect P0? Since this problem goes away after one bad prep and then comes back again a couple of good preps later...do u think the good preps are just because of genetically strong astrocytes (all preps come from the same father tho)!
Thanks in advance. Im out of work here and frustrated, I don't want to read papers for another 2 weeks!!
Mycoplasma, like all organisms with a growth curve, take a while to reach a level where they cause visually obvious alterations. It could be that you have a low level contamination in you medium or some other reagent or location (PBS, pipettes, waterbath, even the animal facility, etc...) which is multiplying once in culture with the astrocytes.
Sora on Fri Feb 25 01:23:05 2011 said:
We routinely extract astrocytes from brain and this protocol has worked for years!!! Recently, a problem started occurring intermittently (we get a bad prep and then a good prep and then a bad one...and so on). When we plate the cells (P0), they look amazing!! We let them grow and they appear really happy. When we split them, they look happy for a couple of days and then...they come off!! The few cells which remain attached look granular and weird....The media doesnt turn turbid even after weeks in culture but the surviving cells just don't divide anymore. We don't routinely test for mycoplasma or viruses, but if it was contamination, why would it only affect the P1 while the P0 are having the time of their lives? Or is it some weird contamination which doesnt affect P0? Since this problem goes away after one bad prep and then comes back again a couple of good preps later...do u think the good preps are just because of genetically strong astrocytes (all preps come from the same father tho)!
Thanks in advance. Im out of work here and frustrated, I don't want to read papers for another 2 weeks!!
It seems unlikely that there are differences between the P0 and P1 cells to explain the problem. What should be checked for differences though is all of the materials. Is there a connection to specific lot numbers? Is it certain operators? Also, how confident are you about your aseptic technique and cleaning procedure between manipulations?