It is crazy that today I was trying to set up my intracellular antibody titration for flow cytometry. Can you please make another video to analyze it too? And talk a little about acqusition on cytometer too?
Dear Aja! Amazing explanation, quick question: if I'm titrating multiple antibodies at once (but in separate tubes) with different fluorochromes each, would it be necesary to make compensantion controls?
Thanks Davis! Because your antibodies are all in separate tubes, compensation controls are not needed (each sample is a single colour is no need to worry about spectral overlap)
On this note however, during the staining process of Blood where you will add a combination of these titrated mABs you will indeed need to account for spectral overlap and thus do a compensation matrixes@@ajarieger_flow
Thanks for reaching out! This gets more challenging. I have generally first titrated the primary with a constant secondary concentration. I then pick 2-4 primary antibody concentrations that look the best and titrate the secondary antibody with each of those.
Be aware that non-specific staining will increase at high secondary antibody concentrations, so it’s important to compare the staining of cells with and without primary antibody present (secondary only control) at each concentration
![FlowJo [TITRATION]](/img/1.gif)
Thank you so much for posting this! You're the best.
Any other requests, let me know! 👍
This soy sauce is fantasticmay I ask what staining buffer u always use? Thank you
Thank you! I have generally used PBS with 2% calf serum for my staining buffer
@@ajarieger_flow is it ok to use just PBS?
@@石一-n4b Generally you will want some kind of protein in there- either serum or BSA. It helps keep your cells happy :)
@@ajarieger_flow thank you so much, I like the way you talk about experiment, which make them vivid and fun
It is crazy that today I was trying to set up my intracellular antibody titration for flow cytometry. Can you please make another video to analyze it too? And talk a little about acqusition on cytometer too?
Great instuctor❤❤❤❤❤
Dear Aja!
Amazing explanation, quick question: if I'm titrating multiple antibodies at once (but in separate tubes) with different fluorochromes each, would it be necesary to make compensantion controls?
Thanks Davis! Because your antibodies are all in separate tubes, compensation controls are not needed (each sample is a single colour is no need to worry about spectral overlap)
On this note however, during the staining process of Blood where you will add a combination of these titrated mABs you will indeed need to account for spectral overlap and thus do a compensation matrixes@@ajarieger_flow
How to do a titration for a primary antibody-unconjugated and secondary biotinilyated antibody?
Thanks for reaching out! This gets more challenging. I have generally first titrated the primary with a constant secondary concentration. I then pick 2-4 primary antibody concentrations that look the best and titrate the secondary antibody with each of those.
Be aware that non-specific staining will increase at high secondary antibody concentrations, so it’s important to compare the staining of cells with and without primary antibody present (secondary only control) at each concentration
@@ajarieger_flow thank you Dr.Rieger.
fascinating
Sweet