Hello,
I am looking for some expertise on culturing MCF7s. I purchased them from ATCC and I have been following all media and culture conditions. Media is EMEM with 10% FBS and 10ug/mL of insulin. When I plated them post thaw, they had a lot of suspension cells with a few settled colonies. I usually spin down the suspension cells and add back to the flask when I am doing a media change. I have passaged them two times now and I still see the floating suspension clumps. I also did trypan blue staining and there are about 34% live cells in the suspension. I feel it shouldn't take this long to settle down. I have now passaged them twice now and I still see the floating colonies. How long do they take to get to a fully adherent monolayer?
Hello, Sushmita!
Please You look a t this protocol:
Article MCF-7 Human Breast Cancer Cells Form Differentiated Microtis...
Cell Culture
MCF-7 cells (HTB-22) [30] were purchased from ATCC (Manassas, VA) and maintained in DMEM-F12 complete media supplemented with 10% fetal bovine serum (FBS), MEM nonessential amino acids, gentamicin and 10μg/mL insulin in a 5%CO2 incubator at 37°C. Media was changed every 2–3 days and cells were passaged when 65–80% confluent. MCF-7 cells were limited to use within the first 10 passages from the original purchased vial from ATCC, to control for genomic drift due to instability. As a comparison, MCF-7 cells purchased from the same lot from ATCC and cultured under the same media conditions were graciously provided by Dr. James Yager of Johns Hopkins University. For 2D cell samples, MCF-7 cells were plated at a density of 300,000 cells/well in 6-well plates and allowed to grow for 3 days. Cells were scraped into Trizol for RNA analysis and stored at -80°C. For imaging studies, all MCF-7 cells were grown on poly-L-lysine coated coverslips for 3 days at a seeding density of 300,000 cells/well in 6 well plates.
When culturing cells, the density of cells is very important, if the density is low, then a monolayer will not form. When you change the environment, it is better not to add the used environment back, always use the new environment. After all, cells grow at the expense of the medium and serum, from which they receive the necessary substances, and if you add the medium used back from the centrifuged cells, then how will the cells grow if they lack nutrients.
Hi Sushmita Nayak
I had a similar problem with HEK293T cells at the end of last year. After doing some research, I washed my cells twice in PBS before passaging. After three passages, there were no more suspension cells and my adhered cells were able to form a healthy monolayer. I would recommend you try the same approach!
If possible, please let me know your findings as I would be interested to know if we had the same issue.
All the best.
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