3T3-L1 cell contamination - (Aug/04/2015 )

Hello everyone,

Lately, I culture my 3T3-L1 passage 14 on a 6-well plate. Two days after I first plate them, in the morning, I observed them and I found out that they were in good condition: cells increase in number, some cells start to differentiate. In the evening, I changed the media, and just in case something goes wrong, I checked them under microscope. I found out that the first well was fine, but the second until the sixth well, the cells deteriorated. Cell confluency drops, and cells became round and detached from each other.

I decided to incubate them, regardless. Two days after, the cells on the first plate grew normally, but those from second to sixth plate were dead. If it's because of incubator or reagent, cells in those walls must have all gone together. I suspect pipetting tips that I use were contaminated. I remembered using the tips from one box, and changed to use tips from the other box, starting from my second wall.

Attached below are photos of the first wall, and the second wall that was contaminated. If it is contamination, what kind of contamination it is? How long does it take to contaminate the cells? I am just an undergraduate student, so I am very new to these lab technique. I am open to any suggestion or advices you may have.

https://www.dropbox.com/sh/k2icq4jgl8lotxa/AAA8RCVLrSZ7xoCbf95500lsa?dl=0

Thank you very much!

Hmmm, it's hard to say from the images, but it doesn't look like contamination. Those lumpy things you can see in one of the images are probably precipitates from FBS. However, the cells don't look happy, they are retracting their processes even in image 1. My guess is that you seeded them at too low a density for them to survive well, or that you have either added something you shouldn't or left something essential out (l-glutamine?)

bob1 on Tue Aug 4 20:33:21 2015 said:

Hmmm, it's hard to say from the images, but it doesn't look like contamination. Those lumpy things you can see in one of the images are probably precipitates from FBS. However, the cells don't look happy, they are retracting their processes even in image 1. My guess is that you seeded them at too low a density for them to survive well, or that you have either added something you shouldn't or left something essential out (l-glutamine?)

Thank you very much for your response.

No. I didn't make any changes with the reagent at all. Or is it because I change the media too frequently? I looked at my lab note just now, and I saw that in less than 48 hours, I changed the media twice already.

By the way, how do I know when I should change the media or passage the cells?

Frequent medium change could in theory remove secreted growth factors that the cells need to survive, and this would be especially prominent at low seeding density. However, I don't know if this is likely to be a problem for 3T3 cells.

According to the ATCC f you let this line get confluent or close to confluent, then they will differentiate into fat cells (irreversible) and as fat cells are terminally differentiated and senescent, which means they won't divide further.

Also note that the ATCC says to use calf serum not FBS!