Hello.
I'm a beginner in flow studies but planning to do in near future.
I'd like to transfect lymph node with mCherry gene in B6 mice, and investigate which cell population has higher mCherry signals using phenotyping.
For phenotyping various cells from single cell suspension, multi-color surface staining is required. And to compensate each signals, I know that FMO control is necessary.
However, I have no idea how to make FMO control for mCherry signals. As you all know, mCherry signal is quite faint so that it should be compensated otherwise false signals can be detected.
Could you give any advice on this?
I'm totally stranger in flow so any advice would be helpful to me.
Thank you!
Jeonghwan
Adriaan is right - having a non-transfected lymph node is the best control to have. I'll add that getting a stronger signal for mCherry is possible, you just need to have in vivo reagents for the job (https://altogen.com/product/peg-liposome-in-vivo-transfection-reagent/). For statistical purposes, its helpful to have a strong signal to begin with, so having higher transfection efficiencies will benefit in the long run.
One of the best controls would be a transfected lymph node without (functional) mCherry, or a non-transfected lymph node - which can be used like an FMO.
Adriaan is right - having a non-transfected lymph node is the best control to have. I'll add that getting a stronger signal for mCherry is possible, you just need to have in vivo reagents for the job (https://altogen.com/product/peg-liposome-in-vivo-transfection-reagent/). For statistical purposes, its helpful to have a strong signal to begin with, so having higher transfection efficiencies will benefit in the long run.
What do you mean by " transfect lymph node with mCherry gene in B6 mice"? How do you do that? And what cells do you wish to phenotype?
Thank you Adriaan and Ted!
I knew that I should include untreated and negative control (from lymph nodes) in my samples but was worried about that there is kinda hinderance between fluorescent signals from a lot of dye-conjugated antibodies. But seemed that my plan would work. Thank you!
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