Hi,



	I was coating 2 FluoroSpot plates at time. Due to some fault in the pipette it took 2 minutes (recommended time in 1 minute maximum) to start washing after adding ethanol. Will this be a problem in the assay?



	Many thanks and best regards!

Hi Ali,



	In the last decade or so Millipore produces FluoroSpot plates where the underdrain has a very resilient design which makes them much less prone to leakage than was the norm maybe 15 years ago. EtOH activation for 2min should not be a problem. 



	The fear is that the excessive etoh activation will cause leakage whereby cell cultures and reagents gets sucked down below the membrane and get trapped within the underdrain attached to the bottom of the plate. This leads to very strange results and a ruined assay. 



	But luckily these problems are a thing of the past.

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Incubated FlouroSpot plates with ethanol more than 1 minute