Morphology of cultured cells - (Sep/23/2013 )
I need suggestion,
I already cultured human osteoblastic cells in 75cm2 flask.
Then I need to compare the morphology of non treated cells and treated.
basically i just take the flask and view under microscope.
how I going to do to check for their morphology? (i want to put the cells on glass slide, then view under microscope , then capture the image)
but need to ensure that the environment is sterile
any staining method that i can use?
or technically support method ?
If you culture your cells on plastic and grow them on glass just to look at the morphology, the morphology might chance just by changing the plastic for glass. Can't you just look at the morphology in the flask you culture them?
the problem is we dont have the microscope that connect directly with camera.
we need to use separated capturing tools such as mobile phone to capture it. so it doesnt give good image.
if you have any method that i can use , please share..
thank you very much for helping
Try plating them on Mat-tek dishes. That way you can view them live on microscope (or fix them).
Or if it has to be on a microscope slide:
Sterilize glass coverslips- dip in 70% EtOH, flame, put in culture dish (6, 12, or 24 well plate, depending on size of coverslips) then UV (take lid off of plate) for 10 min in culture hood
(optional - coat with 50 ug/mL fibronectin in PBS for 1-2 hrs. this is an ECM protein, and it helps cells stick to and spread out on glass. you may not need to, but we use it)
Seed cells on coverslip. Next day, remove culture media and fix (we use 4%PFA)
You can then permeabilize (0.1% triton x-100 or saponin), block (3% BSA in PBS), and stain with markers of your choosing.
Stain in the 6well plate, then when you are done, rinse with H2O (gets rid of salt from PBS) and invert onto glass slide with mounting media.
Hope that helps, let me know if you need any clarification!
You could also grow them on culture slides, which are glass slides with removable plastic chambers on them so that you can grow one or more different cultures on the same slide.
For morphology, I would suggest fixing the cells at defined time points and then staining with a standard dyes such as haemotoxylin and eosin.