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.

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