Hi,
I'm measuring cellular Ca-3 activity with a fluorescence kit (EnzChek Caspase-3 Assay Kit 1) using a Synergy H1 microplate reader.
With this current fluorescent kit the final volume to measure is 100 uL in a well from a 96 well plate.
When measuring the fluorescence I have to fix the gain (fine) but I need the probe height.
What is the best probe height for this sample volume?
Thanks
Does the instrument software have a function for optimizing the probe height with a sample in place?
Dear Jordi,
I think there is a misconception here. The probe height is determined by the plate you are using, not by the assay. You can choose your plate in the read menu of the protocoll editing step. Usually the probe off set for top reads is set to 1 mm in the instrument.
The gain is also something you can determine by you positive (optimal dynamic range) or negative control, by using the auto gain setting in the Gen 5 software (what I would recommend). I think the instrument has a dynamic range of 100 000 RFU. So you can set you positive control to i.e. 80 000 OR your negative control to match i.e. 200 RFU. There by it will make it easy to normalize different plates to each other.
Best Soenke
Dear Sonke,
Thanks a lot for your answer. Indeed, we are now fixing both variables and obtaining some systematic results. Our problem now is that we do not know how to compare the Caspase-3 signal for different concentrations... when the conditions have different number of cells.
Again, thanks.
Dear Adam, We finally got read of the problem and fix this value to a recommended one.
Thanks for your answer
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