Still getting used to flow and up to now we’ve been looking at markers that were strongly positive or negative which was nice and clear cut (perfect intro to flow!), but now we’re in the grey area in between. So, I was wondering if there is a standard cutoff for isotype controls- e.g. less than X % positive, or on a dotblot/histogram are you supposed to set the marker to exclude all the cells in the isotype control- sometimes there are a few scattered up higher than all the rest, well away from the cluster. When there is not much of a positive shift in the analytical sample for detection of rare events,moving the isotype control setting from 0.7% to 1.2% can result in a 5-10% change in the reading for the analytical sample. Obviously simple subraction would not account for this. I know this is a basic question and any advice would be greatly appreciated- even just direction to a good paper or book to clarify this.
Isotype control gating
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