HCT 116 cells were obtained from NCCS, Pune, India. The cells were seeded in T25 flask in Mc Coy's complete media with 10% FBS and 1% PenStrep antibiotic and incubated at 5% CO2 and 37 degree celcius. When the cells were cultured in 96 well plate (5000 cells/ well) and incubated for 24 hours or more, the spreading of the cells were not observed. In T25 flask the cells grew well. The cells in the plate were attached but round in shape. What could be the reason that the cells were not spreading?
Are your cells at the same density in T25 and 96w? If yes; is you 96w plate of TC quality? Another possiblity is that the tissue culture surface/cell ratio is very different in the two cultures, ie. you have less surface/cell in the 96w plate, you could try 10 fold less density in the 96w plate. Airation is a problem sometimes in 96w plates, you could see that also from the color of the medium in the perimeter and in the centrum, it should be the same. Lastly, mycoplasma infection could cause changes in the way cells are growing, you could check for mycoplasma infection.
could be the coating of the plate, static energy (you can solve that by putting plate in the freezer for a few hours.)
Thanks Johan, well HeLa cells are growing really well in the same 96 well plate acquired from NEST but the problem is with HCT 116.
Can you explain what exactly you are suggesting here?
Should we put the fresh plate in the freezer (4 degree/-20 degree) before using?
Is possible that the quality of the plate is notgood for cell culture. The base of the weell should be flat and not round. I consider that the cell density/well is high.
The plate's bottom is flat and we have procured it from NEST so I guess quality is not a problem.
A number of probabilities are there:
Firstly, Plate surface is not charged, as charged surface is a requirement for binding of cells to the surface.
secondly, composition of media is not appropriate, if it is too old then add some nutrients like amino acids and fetal bovine serum or increase their composition.
another problem can be the type of media you are using. check the type of media required to grow your cell lines. normally HCT-116 are grown in DMEM or RPMI media, sometimes MacCoy is also added.
check the environment in the incubator, like temperature, carbon dioxide and humidity.
Himanshu the only problem is cells not growing in 96 well plate, in T75 flask they are growing normally what ever points you have included are same for growth in the flask.
I would check the date and lot of the plates you are using and make sure they were made for cell culture and not for PCR etc. We have had these issues before with certain lots, and with batches older than 2 years. Call the manufacturer with the lot number and make sure they haven't had any complaints.
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