Dear all,
I am trying to count alive spore from dead one after incubation with macrophage by using flow cytometer,
Does anybody has an idea, how can I select suitable label to my spores and dye?
My spores already transformed with GPF plasmid and confirmed the expression of GFP,
Regards
Ayat
Hi Ayat
You could use a viability dye suitable for your spores (like Propidium Iodide or 7AAD) e check the GFPpos PIneg. Those will be your live spores.
Thank you Andre Fernandes for your answer,
Do you mean label my spores with this dye before incubation? but how can I recognise the live spores from dead? I want to count killing by macrophage to my filamentous fungi spores, I did killing assay by counting CFU and I want use flow cytometer for more accurate results
And what do you mean of e check the GFPpos PIneg??
Hi Ayat
You should label your spores after macrophage processing. Dyes like PI (propidium iodide) and 7AAD are only internalized by dead cells. In that case live spores will be PI negative (PIneg) and dead spores will be PI positive (PIpos). You should conduct your experiment and lyse your macrophages as you would for CFU but instead of plating you conduct the staining for flow cytometry. If you gate first on GFPpos cells you will only analyze spores and then if you gate on PIneg you will have the live spores.
Dear Andre Fernandes
I am working with filamentous fungi, my strains transformed with GFP
I should use spores of strains transformed with GFP plasmid
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