Spectral Cytometry: unmixing autofluorescence
Description
Data prepared for the 2024 Babraham Spectral Symposium. Mouse samples stained with an unoptimized panel intended to create difficulties in spectral unmixing. Acquired on a 5-laser Cytek Aurora. Bead (UltraComp Plus) and cell controls are provided for single colour stains. Unstained samples of spleen, lung and brain are provided for autofluorescence extraction options.
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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.