Hii!
So I designed for my next experiments a quite complex panel and I have a few questions about how better proceed for treating my Abs and building my compensation.
So I have a Fixable Live/Dead Near IR she in the same channel that my lineage cocktail that I will gate out. And I have intranuclear markers I stain for too.
So my questions are as follow:
1- Is it correct to first titrate my Abs in cells and then use beads for the compensation using the selected concentrations?
2- For compensating the channel of my Live/Dead dye and lineage markers, should I use one of the markers or the dye?
3- As I will titrate first and I will be able to see the signal in cells, should I keep similar voltage I have set during that titration for the recording od the compensation beads? Even if it does not have the best signal to ratio ?
4- As I have intranuclear staining for some of the markers, do I need to compensate that ones with cells that were also fix and permeabilized?
As you see, I have many qiestions! Hope you can help with them.
Thank you very much in advance!
Alba
Choosing your colors depends on your cytometer. I recommend using Fluorofinder (fluorofinder.com). Use the Chrome web browser. For some reason, this website is extremely responsive on Chromebooks, just FYI. This will help you to choose your colors while minimizing spectral overlap and maximizing their compatibility with the specific instrument. This is important because you do not want your colors to interfere with eachother. If you need to add your cytometer’s configuration to the website, just message support, and they will help you to walk through the process.
Once you have your antibodies, you should individually titrate them. Set up some positive controls, and stain your cells at like five differenr concentrations. Run your cells at a consistent low speed on the cytometer (highly concentrated sample but no faster than around 2,000 events per second), and then calculate the staining index using an equation that you can find online. You will probably find that you can use less antibody than is recommended by the manufacturer.
Finally, stain your single-stained controls, FMOs, controls, experimental samples, and do not forget your single-stained controls, hop on the instrument, optimize your voltages based on your fully stained sample and refining them if your single-stained controls go off scale.
Sorry, for multiple posts. The developer for the iOS app seems to have implemented the wrong kind of text field, so I have no ability to scroll (also, there is no autocorrect or grammar-check)
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining