3T3-L1 cells, is this a bacterial contamination or a cross contamination with different cell line? or something else?

14 February 2021 3 4K Report

Hi,

I am experiencing some difficulties with 3T3-L1 cells culturing recently. I am working with 3T3-L1 cells for almost 4 years now and have seen bacterial contaminations/ unhealthy cells and usually have figured out the problem. But in this situation, I am unable to figure out what went wrong.

Any constructive comments and suggestions are highly appreciated.

*After 2 days of thawing the cells, the cells were about 70% (Fig 1 and 1a). and I passaged the cells into new dishes.

* when I checked the cells after 2 days of passaging, they appeared in a bad condition.

Procedure used for passaging:

* cells were washed with 1X HBSS

* added 1 ml of 1X trypsin

* kept in incubator for 2 minutes

* collected the cells by 3 ml of media

* centrifuged it at 450g for 5 min at RT

* discarded the supernatant

* added new media and divided in into 4 new plates.

There were some brown adipocyte cell lines in the same incubator. Can it be a cross contamination? But the cells seemed to be fine before passaging.

I have attached the images of the cells before (Fig 1 and 1a) and after passaging (Fig 2 and 2a).

I will thaw cells again from another vial for my experiments but I wanted to know what might be the possible reasons for this condition. I am new in a lab and the procedure is slightly different from what I have been used to.

Thank you again.

Miriane de Oliveira

Hello how are you? I suggest that you do a test to check the bacterial contamination of each of the reagents that you used to pass to other plates. With this you can identify which reagent is contaminated and discard it.

María Luna-Luna

Hello. I don't see the bacterial contamination in your photos. But you could check your medium. On the other hand, some cells are sensible with the type of trypsin. I don't know if your cells were viable the next day (day one after of the trypsin).

Shalom Sara Thomas

Thank you for the replies. I think the trypsin I used was having some problems. When I checked the HBSS and BS media it didn't show any contamination.

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