Like most people, I rarely use all 96 wells on a 96 well plate. I typically avoid the wells at the edge, which means only using 60 wells and sometimes I use as few as 16. I load typically load the unused wells with water or PBS to limit evaporation from the cell culture wells. However, I had one colleague who insisted on filling unused wells with media, because he believed the gas exchange would be perturbed if a media well was surrounded by PBS/water wells. Does anyone know if there's a scientific basis for this practice?
John
I think your colleague has a little exaggerated the problem. Depending on your protocol, if you coat your plate for one hour, then incubate your 1st Ab for one hour, and incubate your 2nd Ab for one hour, and then develop your plate for reading, the variations you mentioned above (media, water, or PBS) don't make much difference.
If you culture the cells over 24 hours, the media is best choice. If only several hours, PBS is OK.
Adding PBS or water to the unused wells should be fine, and you don't have to fill the unused wells either. If the incubator is properly humidified, there should be little evaporation anyway.
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