AutoSpectral: ID7000 40C examples
- olivertburton
- 1 day ago
- 2 min read
In this post, we looked at some data from OMIP-102 run on the FACSDiscover S8 and unmixed using AutoSpectral. Today, let's look at some 40-colour data from a 5-laser Sony ID7000. The panel here was developed by Stephanie Humblet-Baron's group at KU Leuven for a human immune phenotyping platform, and it covers a diverse array of cell types present in peripheral blood with some in-depth phenotyping of B and T cells.
A couple of notes before we look at the data:
This panel has a bit of an unmixing hotspot in the violet around 500nm. This is because the panel has BV480, StarBright Violet 515, BV510, StarBright UV445, BUV496 and FITC, which is pushing what this ID7000 can do.
The default for this panel is not to do autofluorescence extraction. Adding even a single autofluorescence parameter with the standard weighted least squares approach causes an explosion of the above hotspot.
The comparison data file here for the unmixing on the Sony software is done with bead controls, except for the viability dye. This is the recommendation from Sony, and I personally found it challenging to unmix the data in the Sony software using the cells stained with the markers in this panel. I don't have a ton of experience on the ID7000, so that may be a skill issue. So, rather than show something that would be less than useful for a comparison, I'm showing the best unmixing I could get on the Sony, which is also representative of what most people can achieve, I think.
All the unmixing here is done using weighted least squares as the base algorithm.
Gating is on scatter for lymphocytes + monocytes, single cells and viability (excluding dead cells).
