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

After doing what was recommended, we got some robust results.

What I do recommend is to normalize the signal per number of cells AND repeat the experiment to minimize day to day and experiment to experiment errors.

We are happy to use around 7 samples per condition with high repeatability.