How do I gate a population of nuclei from debris in a FSC vs. SSC plot, or any other plot for that matter? I am analyzing a live single nuclei suspension from rat brain tissue which are stained with a Höchst DNA dye and the neuronal marker NeuN.
Hi Johanna,
From what I understand, gating isolated nuclei in a FSC/SSC plot is very difficult, as nuclei will vary in size depending on their current cell cycle and they also tend to aggregate when they are in isolated conditions.
In order to gate isolated nuclei properly, I suggest comparing a histogram of your NeuN data overlayed onto an isotype control. This will allow you to determine the "true positive" nuclei from just nonspecifically stained debris. I have linked a protocol that may be helpful below from Hengstschläger, et al.
http://www.nature.com/nprot/journal/v8/n3/full/nprot.2013.011.html
Hello Johanna,
isolated nuclei from rat brain, if the tissue is fresh, should be easy to gate from a FSC/SSC diagram. Since brain cells generally do not cycle, nuclei should all be the same size/structure and stand out in the scatter plots. If the Hoechst stain really works (and it works best on unfixed nuclei), then it should enable to combine any scatter signal with the Hoechst fluorescence intensity signal, which should be proportional (approx) to the DNA content of the nuclei. Comparison eg with chicken erythrocytes should give the absolute DNA content of the mouse brain nuclei. The neuronal marker should be labelled with a dye not interfereing with the Hoechst dye, so a number of two parameter plots can be obtained: FSC/SSC; Ho/FSC; Ho/Marker etc. Depends on the brand and type of your FCM.
Hope this helps, but feel free to ask for more.
Harrie
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