Morag MRKO flow data.prism

Flow cytometry methods

Immune cells were harvested by passing spleens through a 70mm filter, after which 20 ml of PBS was added and the cell suspension spun (500g, 5 min, 4°C). Red blood cells were lysed and white blood cells subsequently stained with a surface antibody cocktail [CD45-PB (Biolegend Cat#103126), Gr1-PerCP (BD Biosciences Cat#552093), CD115-BV605 (Biolegend Cat#133517), CD3-FITC (Biolegend Cat#152304), CD19-BUV395 (Biolegend Cat#563557), F4/80-PE-Cy7 (Biolegend Cat#123114)] for 30 min in the dark on ice. Next, 500 ml of FACS buffer was added to samples and samples were spun (500g, 3 min). After aspirating the supernatant, 100 ml of Cytofix/Cytoperm buffer (BD Biosciences Cat#554722) was added to cells. After a 30 min incubation on ice, 100 ml of Permwash buffer (BD Biosciences Cat#554723) was added and cells were spun (500g, 3 min). Cells were stained with an intracellular antibody cocktail [IL-1b-PE (ebioscience Cat#12-7114-82), IL-10-APC (BD pharmingen Cat#554468)] for 60 min in the dark on ice. After which 500 ml of Permwash buffer was added and cells were spun (500g, 3 min). After aspirating the supernatant, cells were resuspended in FACS buffer and marker expression determined on an LSR Fortessa (BD Biosciences) using FACS Diva software. Compensation controls were run in form of cell samples stained with single antibodies, and, in the case of the cytokine antibodies, because cytokines are typically lowly expressed in un-stimulated cells, compensation beads (Ultracomp beads, Invitrogen Cat#01-2222-42). Cells were gated as follows: after initial gating on FSC v SSC, doublets were excluded based on SSC-A v SSC-H and leukocytes identified as CD45⁺ cells. From a plot of CD115 v Gr1, neutrophils (CD115^- Gr1^high), Ly6C high monocytes (CD115^highGr1^high) and Ly6C low monocytes (CD115^high Gr1^low) were identified. From a population of CD115^intermediateGr1^intermediate expressing cells, macrophages (F4/80^high) and eosinophils (SSC^high F4/80^-) were identified. From the CD115^-Gr1^- population, T cells and B cells were identified based on CD3 and CD19 expression. Expression of IL-1β and IL-10 was quantified using the mean fluorescence intensity (MFI).