The hormone binding reaction was initiated by addition of fluorescent tagged natural ligand to each in 6 well plate (4 to 5 lac cells/ per well 6 well plate). Incubations were performed in triplicate for 30 min at room temperature. After incubation ,cells were scrapped by cell scrapper. Cell suspension was centrifuged 1000g 10 min at 4 o C, then discarded the supernatant and cells were fixed in 4 % PFA over ice for one hour. After one hour PFA was discarded after centrifugation and cells were suspended in facs buffer and analyzed according to convenience in few days. I have doubt here about washings and fixation, whether they are essential or it can be avoided? If essential when i should perform
Why do you scrape the cells? Can't you lift them enzyamatically (dispase )? You should also check that the ligand stay on at every stage of your isolation. You should also be able to do the assay by florescent image analysis of living or fixed cells but you might have to culture them on glass cover slips.
Dear Slobodon Sir, cells doesn't detach very easily with enzymatic as well as non-enzymatic way. Alternatively, I have tried to do the above experiment in another way. The cell line I am using is clone 9. In alternative way, I am detaching the cells by PBS -EDTA. And incubating the cell suspension in incomplete cell culture media with desire fluorescent ligand at 37 degree 30 min. After 30 min, I centrifuge cell function rapidly for short duration, 15000 g 30 sec. Then discarded the Supernatant. The cell pellet was immediately suspended in PBS and analysed cells by facs. I am getting signal. And I feel this alternative method little cumbersome. Whether this alternative method I have used is scientifically correct or there is need to add some steps? Thanking you.
I still think the simplest way to do this is on attached cell cultures using image analysis. Hopefully you are aware that FACS will count cell fragments as well as cells. Detaching with PBS/EDTA is preferable to scraping but you dont need to label by incubation in the incomplete culture medium.
Dear Slobodan Sir, I totally agree with you that FACS will count cell fragments as well as cells. In order to avoid cell debris and large particles, we are selecting the uniform population with SSC vs FSC as per the control cells. Thanks for your valuable suggestion.
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