Hi,
I stained bivalves haemocytes with PI (final concentration 10ug/mL) , SYBR Green (final concentration 1x and 10x) and PI+SYBR Green. The concentration I used are the ones commonly used in the literature but the flow cytometer (NovoCyte Advanyeon) cannot detect the fluorescence which is probably too high (in the cytogram it says events are out-scale or saturated).
I do not understand why I have this issues.
Can someone please have any suggestions?
Thanks in advance
Elisabetta
If your events are too bright, you can either decrease the voltage of the PMT detectors or decrease the gain setting or both. I have never worked with SYBR Green, but I know that PI is a very bright stain for dead cells, and I have to reduce the voltage of the PI detector to get it on scale.
If your cytometer says "events are out of the graph", it means either you need to decrease the voltage/gain or decrease dye concentration.
Try one thing at a time, you will definitely get events.
If most of your cells are alive, then no uptake is taking place. Treat an aliquot with 0.5% SDS and run it. It should glow.
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