Cs t calibration beads

All flow cytometry experiments were performed at the O2 Flow Facility at Amsterdam UMC (Netherlands) using an X20 Fortessa flow cytometer (BD Biosciences). The cytometer was daily calibrated using CS&T calibration beads (BD Bioscience), and all samples in the study were measured with the same lot number of CS&T calibration beads. For acquisition, all samples were filtered using a 70 µm cell strainer, resuspended in 250 µL and acquisition was performed by a plate loader set at 1.0 µL/s acquisition speed. Flow cytometry data were analyzed first using FlowJo analysis software. First, files were compensated using UltraComp eBeads (Thermo Fisher) microspheres labeled with the appropriate fluorochrome-labeled antibodies. Compensation was additionally verified using FMO controls for every single fluorochrome for every tissue type (equally pooled per group) on experimental samples. First, gating was performed on a stable flow (time vs cell count), subsequently on viability dye-negative/lin-negative/CD45-positive cells and finally on single cells (FSC-A/FSC-H). The resulting cells of all individual samples were concatenated per tissue type and exported per experimental group into an Flow Cytometry Standard (FCS) file and uploaded to the Cytobank online analysis platform (https://www.cytobank.org/) for tSNE and CITRUS analysis.

Following cell staining, samples were acquired in a BD LSR Fortessa SORP cytometer. The FACS tubes were acquired completely. Spectral overlap compensation between all channels was done automatically by the BD FACSDiva software v8.0.1 (BD Biosciences, Franklin Lakes, USA, New Jersey) using single-color controls. Comparable day-to-day performance of the cytometer was ascertained by running CS&T calibration beads (BD) weekly. In addition, for standardization of instrument settings longitudinally, an 8-peak Rainbow Compensation Particles Set (BD) was used prior to every experiment, and photomultiplier tubes (PMTs) voltages were adjusted if needed. Flow cytometry data were analyzed using FlowJo v10.8.1 (TreeStar, Portland, USA, OR). The FlowJo boolean gating tool was used to create the co-expression profiles.