I'm planning to do flow cytometry to check the purity of a B cell fraction sorted from bone marrow with magnetic beads.
I will stain the B cell fraction with an APC-Ab anti CD19 and I will also use PI to check viability.
Do I need to do any compensation between APC and PI?
The answer should be: you don't know until you run your own controls. Prepare single stained compensation controls (APC Ab bound to comp beads and permeabilized cells stained with PI), compensate with software via the machine or FLowJo and look at the matrix to see the fluorescence subtraction. You can find more about compensation controls on the Purdue Cytometry List.
Hi, Marilina
As already stated by Raphael, you shouldn't have any issues regarding compensation between these two fluorescences. Specially if you have access to an instrument with laser-specific set of detectors. PI is mainly excited by a blue laser while APC is excited by a red one.
Kind regards,
R.
Hi,
I'm agree : no compensation. But take care if you are using a flow cytometer such as FC500 (Beckman coulter) : the 2 lasers are colinear and you will have problems if you use APC and PI at the same time...
The answer should be: you don't know until you run your own controls. Prepare single stained compensation controls (APC Ab bound to comp beads and permeabilized cells stained with PI), compensate with software via the machine or FLowJo and look at the matrix to see the fluorescence subtraction. You can find more about compensation controls on the Purdue Cytometry List.
As said, PI works fine and 7-AAD can be an alternative as it is very similar to PI and in theory not excited by the 633 nm laser. Depending on your machine and your combination, you can also use dyes excited by other lasers. Sytox Blue is excited by the 405 nm laser and its emission is far away from APC.
Hi,
there shouldn´t be a problem, because there are around 18,000 detectable molecules and you do have a strong color; but in theory: still it is another laser, there could be some slight compensation due to an excitation of PI by the red laser of ~ 6%. 7AAD is a good alternative like Stephane Mancini wrote and is absolutely not excited by the red laser.
other live/dead...
http://www.lifetechnologies.com/de/de/home/brands/molecular-probes/key-molecular-probes-products/live-dead-viability-brand-page/live-dead-fixable-dead-cell-stains.html?s_kwcid=AL!3652!3!32673457224!b!!g!!dead%20cells%20flow%20cytometry&ef_id=V1FQG70uY8AAAAbx:20131216161200:s
http://info.ebioscience.com/bid/58846/Flow-Cytometry-Discriminate-Between-Live-and-Dead-Cells
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