Tissue autofluorescence (high dimensional)
Description
Mouse tissue samples from spleen, lung and liver. Stained with lots of random markers to identify and measure spillover artefacts associated with autofluorescence.
Files
Steps to reproduce
Tissues from mice on the C57BL/6J background were prepared as in https://www.cell.com/immunity/fulltext/S1074-7613(24)00277-2. In brief, the spleen was crushed between glass slides and the other tissues were digested with collagenase prior to Percoll density enrichment. Prior to surface staining, cells were blocked with 5% mouse serum and 5% rat serum in 100ul PBS + 2.5% FCS + 2mM EDTA for 15min at 4C. Cells were stained for 1hr at 4C in 100ul PBS + 2.5% FCS + 2mM EDTA. After two washes, the cells were fixed with the eBioscience Foxp3 Fix/perm kit for 30min at 4C. The cells were then washed twice with permeabilization buffer. The cells were then blocked again with 5% rat serum in 100ul PBS + 2.5% FCS + 2mM EDTA for 15min at 4C. Cells were then stained overnight at 4C in permeabilization buffer. The cells were then washed twice with permeabilization buffer and resuspended in PBS + 2.5% FCS + 2mM EDTA. Tandem stabilizer was included during staining steps and in the final resuspension. Antibodies used and the dilution factors are indicated in "20250218 unmixing speed panel.xlsx". Samples were acquired on a 5-laser Cytek Aurora using CytekAssaySettings adjusted only for scatter settings. Both BioLegend compensation beads and cells are provided as single-color controls for unmixing. The samples were unmixed entirely with cells in the data provided.
Institutions
- University of Cambridge