Hi there,
I'm doing spheroid culture on agarose-coated well plates (flat bottom). How can I ensure or provide some sort of proof that the coating is always "the same"? Any idea? I thought about measuring coating thickness with a scaled probe. Maybe adding a dye to the agarose and measuring absoprtion would also be an option. However, naturally the coating is never flat like the bottom of the well, but curves like a meniscus. Does anyone have any experience or tips to help me with this?
Maybe gelling agarose under centrifugation condition could help reduce meniscus.
Form a Statistical side, you need to repeat/ perform your measurements at least 6 times 2 and perform agreement analysis of the results
I agree with the centrifugation method. You place a fixed volume per well, spin at low RPM using a swinging bucket rotor, this will promote even distribution during the gellin period. if you use the same RPM and time of spin each time, it should be very reproducible.
About the curve like a meniscus, is it not supposed to form a curve? I think the idea about using agarose coated plates is precisely to avoid cells adhesion and also to form a concave surface for liquid overlay spheroid culturing.
In plate spectrophotometers from TECAN you can find function of multiple measurements per well (f.e. 5x5 point, square or circus). This kind of measurement of agarose layer with any suitable dye could reflect uniformity of created coating.
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