AutoSpectral: Difficult cell controls, unmixed better
Description
Data prepared for the 2024 Babraham Spectral Symposium. Unmixing in SpectroFlo software. The fully stained data are the same as in AutoSpectral: Difficult cell controls. In this set, much more effort has been made to optimize the unmixing in SpectroFlo. Mouse samples stained with an unoptimized panel intended to create difficulties in spectral unmixing. Acquired on a 5-laser Cytek Aurora. Unstained samples of spleen, lung and brain are provided for autofluorescence extraction options.
Files
Steps to reproduce
Isolate cells from tissues (see https://www.cell.com/immunity/fulltext/S1074-7613(24)00277-2?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1074761324002772%3Fshowall%3Dtrue for details). Plate 2 million cells in V-bottom 96-well plate. Block with 2.4G2 hybridoma supernatant 20min at 4C. Wash with FACS buffer (PBS 2.5% FCS 2mM EDTA). Stain 1hr with antibodies at indicated dilutions. Wash with FACS buffer (PBS 2.5% FCS 2mM EDTA). Fix 30min with formalin. Resuspend in FACS buffer. Prepare single-stained cell controls as per above protocol. Prepare UltraComp Plus bead controls in PBS.
Institutions
Categories
Funding
European Research Council
Tissue-Treg
Wellcome Trust
Wellcome Investigator Award, 222442/A/21/Z
UK Research and Innovation
MR/Y004450/1 (IMMPROVE)
European Research Council
No 874707 (EXIMIOUS)