Hallo everybody, The users here are encouraged to follow protocols from "Current Protocols in Cytometry" whenever possible. Recently we've started doing a lot of DNA staining and have noticed variation in fluorescence between similar samples when utilising PI. On scrutinising the given protocol in CPC, it says you can have a cell suspension of 1 million to 10 million cells for PI. For Dapi it says 100 thousand to 1 million, and for Hoechst a suspension of 1 million. Somewhere in the past I learnt that you use a constant number of cells depending on the protocol. I take it it should mean you can stain up to a certain concentration of cells but omits to explain that the cell number per sample should not vary. However, if you can vary the cell concentration to such an extent surely the A-T bases are no longer saturated and will give sub-optimal staining. Oui??? In the case of Hoeschst where the users go by the 1 million cell conc. we do not see the peaks shifting up and down between samples. Regards Ann Merry Xmas
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