Great question! Standarisation between experiments or across machines/sites can be important as you want the cytometer to be operating at the same sensitivity each time you run samples. Some manufacturers can do this automatically eg BD uses Application settings which can be linked to the CS&T QC settings.However, you can do this yourself - once you have set up the machine for the initial experiment, you can run a bead, or beads, and note the median fluoescence intensity in the relevant channels. The next time you run the experiment, run the same beads and adjust the voltage/gain if necessary to bring the bead to the same intensity, in that way you will be running at the same sesnitivity and it will be easier to compare MFI vales of your cells.
How do you know what antibody titre to choose for your specific experiment? when would you use your saturating titre and otherwise the separating titre?
You want to find the minimum amount of antibody that will give you maximum separation. The less antibody you use, the more money you save and you also reduce the likelihood of any unspecific staining. But you need enough to maximize the separation between negative and positive populations for that specific marker you're measuring. Sometimes, however, if you already have a really good separation, and you are not looking at differences in levels of expression, you can reduce the amount of antibody to minimize any potential spillover signal of that conjugated fluor in other channels. You still end end up a good separation, but you reduced the amount of signal that can potentially spillover into other channels.
The FITC channel as well as the PE channel of the 488 laser are typically good channels for AF. There are also channels in the 450-550nm emission range off the violet or UV channels that will also be good to check AF. This obviously depends on the AF spectral signature of your cells, so trying out a few channels where you see an obvious diagonal shape in the unstained population(s) will do the trick. But definitely not random :)
![FlowJo [TITRATION]](http://i.ytimg.com/vi/XQTGms5BkeA/mqdefault.jpg)
Thank you for the video! I really appreciate this content.
thank you for this!
Could please guide on MFI tracking across experiments using the same panel. So that MFI values are comparable across the experiments.
Great question! Standarisation between experiments or across machines/sites can be important as you want the cytometer to be operating at the same sensitivity each time you run samples. Some manufacturers can do this automatically eg BD uses Application settings which can be linked to the CS&T QC settings.However, you can do this yourself - once you have set up the machine for the initial experiment, you can run a bead, or beads, and note the median fluoescence intensity in the relevant channels. The next time you run the experiment, run the same beads and adjust the voltage/gain if necessary to bring the bead to the same intensity, in that way you will be running at the same sesnitivity and it will be easier to compare MFI vales of your cells.
How do you know what antibody titre to choose for your specific experiment? when would you use your saturating titre and otherwise the separating titre?
You want to find the minimum amount of antibody that will give you maximum separation. The less antibody you use, the more money you save and you also reduce the likelihood of any unspecific staining. But you need enough to maximize the separation between negative and positive populations for that specific marker you're measuring. Sometimes, however, if you already have a really good separation, and you are not looking at differences in levels of expression, you can reduce the amount of antibody to minimize any potential spillover signal of that conjugated fluor in other channels. You still end end up a good separation, but you reduced the amount of signal that can potentially spillover into other channels.
Is the autofluorescence channel chosen at random or there is a specific reason for choosing FITC in this specific experiment?
The FITC channel as well as the PE channel of the 488 laser are typically good channels for AF. There are also channels in the 450-550nm emission range off the violet or UV channels that will also be good to check AF. This obviously depends on the AF spectral signature of your cells, so trying out a few channels where you see an obvious diagonal shape in the unstained population(s) will do the trick. But definitely not random :)