Can someone suggest an optimum cell seeding density (& media volume) for seeding isolated nenatal rat cardiomyocytes on a 25mm coverslip? I have fibronectin micopatterns on the coverslips for cell attachment and PLL-g-PEG for non-adhesion, but am unable to attain good confluence.
Hi...I suggest you to take 200 μl of an aqueous fibronectin solution (50 μg/ml) was applied to each cell culture insert.After 2 h incubation at room temperature, the fibronectin solution was removed and the cell culture inserts were rinsed three times with PBS. Then 5x104 or 4x105 seeding density you can use for maximum confluence.
All the best..
Thank you, I shall try it this time. Also, would you suggest having about 4x10^5 cells in about 1.5 ml media or have them more concentrated, say in 250 ul meida - so they would attach only on the patterns. These are freshly isolated primary cultures.
My patterns is an 3x3 array of rectangles ( 900 um by 500 um) - I use microcontact printing to transfer the proteins.
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