Hello All,
I am trying to do a simple fluorescence plus Giemsa stain/acridine orange differential stain for chromosomes. I have had great success in the past using MEF/CHO/and various cancer cell lines, however when I switched over to MCF10a's I have had no luck. I am using a 3 mg/ml BrdU in PBS stock and a 1:1000 dilution for a 10 uM concentration in the growth media. I am incubating the log phase cells in the 10uM BrdU for 32-48 hours prior to adding colcemid for an addition 4 hours before collecting the chromosomes. Has anyone else encountered trouble with MCF10a's incorporating BrdU (even flash labeling or for proliferation assay), if so how were you able to overcome this issue?
Thanks
AS you know MCF10A is a normal human breast cell line requrires special media . tThe cultivation is different from the other cells .
The BrdU is added to the specialized MCF10a media, we have had excellent success using mcf10a's in the lab for several years. This is first time we are using BrdU incorporation though
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