AutoSpectral: Full Workflow Example
Description
Mouse tissue samples from spleen, lung and liver. Stained with key cell type-defining markers with well separated fluorophores to identify and measure spillover artefacts associated with autofluorescence.
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Steps to reproduce
Tissues 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. 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 with the following: CD3-APC 1:200, CD4-BV421 1:500, F4/80 PE/Cy7 1:200, CD11b-BUV805 1:200, Siglec F-PE 1:400, CD45-BUV395 1:500, fixable viability dye eFluor780 1:4000. 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 with cells in the data provided.