Ficoll paque

The EAI assay was performed as described in detail elsewhere [9 (link)]. In brief, erythrocytes obtained from Balb/cJRj mice were purified using a Ficoll gradient (Ficoll-Paque, VWR, Cat#17-1440-02) and were stored for up to 2 weeks in DMEM (Gibco, Cat#61965) containing 10% FCS FCS (Amimed, Cat#2-01F30I) at 4°C prior to use.
Serial dilutions of sera were performed in 96-well U-bottom plates (Greiner, Cat#650161) in DMEM containing 10% FCS. 5x10⁵ cfu B. taylorii expressing GFP were added per well and the plates were incubated at 35°C, 5% CO₂ for 1h prior to the addition of 10⁶ red blood cells (multiplicity of infection, MOI = 0.5) in 100 μl DMEM containing 10% FCS. The next day, the supernatant was removed, the red blood cells were fixed using 1% PFA (EMS, Cat#EMS-15710) and 0.2% gluturaldehyde (EMS, Cat#16020) in PBS (BioConcept, Cat#3-05F29-I) for 10 min at 4°C in the dark. After quenching with 2% FCS in PBS, the cells were analysed for GFP signal by Flow Cytometry (BD Canto II using HTS autosampler).

PBMCs (peripheral blood mononuclear cells) were isolated by density gradient centrifugation using Ficoll paque (VWR, Austria) and frozen (-196°C) for analysis at a later time point. Analysis of T-cell subpopulations was performed by flow cytometry (FACSCANTO II, BD, United States) using specific labeled antibodies against CD3 (APCCy7), CD4 (APC), CD8 (BV421 or PECy7), CD45RO (PE), CCR7 (FITC), CD16 (FITC), CD19 (PE), and CD25 (PE) all from BD/Europe, Biozym/Germany or eBioscience/Austria and the viability dye 7-AAD (Sigma/Germany). At first, lymphocytes were gated on a forward scatter/side scatter (FSC/SSC) dot plot following gating on living cells and CD3/CD4 or CD3/CD8. Combinations of surface markers were classified to define the respective immune cell subpopulations as stated in Table 1 and Figure 1. For quality control, we followed standard procedures of the lab, which includes determination of the % bright bead robust CV for the measurements according to the manufacturer’s instructions.

Peripheral blood was collected from healthy adult volunteers according to protocols approved by the ethics board of The Francis Crick Institute. Peripheral blood mononuclear cells (PBMCs) were freshly isolated by density gradient centrifugation in Ficoll-Paque (VWR) and CD8⁺ T cells isolated by negative selection using a CD8⁺ T Cell Isolation Kit (Miltenyi Biotec).

Venous blood was collected in sterile 10 mL EDTA and 8 mL serum BD Vacutainer tubes (Becton Dickinson) and processed within 1–4 hours. Isolation of PBMCs was performed on freshly collected blood by density centrifugation over Ficoll-Paque (VWR) as described previously (70 (link)). Monocytes were isolated by magnetic-activated cell sorting using negative bead selection with the Pan Monocyte Isolation Kit (Miltenyi Biotec) according to manufacturer’s instructions. Cell counts, cell purity, and composition were evaluated by XN-450 hematology analyzer (Sysmex Corporation). Fresh isolated cells (monocytes 1 × 10⁵ cells per well, PBMCs 5 × 10⁵ cells per well) were incubated with different bacterial, fungal, and viral stimuli (Supplemental Table 2) at 37°C and 5% CO₂ for either 24 hours or 7 days. For the 7-day stimulation, 10% human pooled serum was added to the wells. IL-1β, IL-6, IL-1Ra, IL-10, and TNF-α were determined in the supernatants of the 24-hour PBMC or monocyte stimulation experiments, using ELISAs (Duoset ELISA, R&D Systems). IL-17, IL-22, and IFN-γ were measured after the 7-day stimulation of PBMCs (PeliKine Compact or R&D Systems). For intracellular cytokine measurements of active IL-1β and pro–IL-1β (Quantikine, R&D Systems), cell pellets were lysed using Triton X-100 (MilliporeSigma).

Peripheral blood monocytes were isolated from human blood using 15 ml Ficoll-Paque (VWR) centrifugation gradient. Centrifugation was performed at 1000×g for 1 min, with added 9 ml heparin and filled to 50 ml with PBS. Centrifugation was then repeated at 1000×g for 10 min, discarding the upper plasma layer and collecting the PBMC layer. The cells were washed two times with PBS and centrifuged at 350×g for 10 min. The supernatant was discarded and cultured in 5 ml basal Iscove’s medium supplemented with 1% Pen/Strep, 10% FCS (Biochtrom, Berlin, Germany) and subsequently incubated at 37°C and 5% CO₂ for 2 hr before seeded and imaged on an 18 mm diameter microscope cover glass (VWR) in PBS containing Ca²⁺ (Gibco) at room temperature.

Buffy coats were procured from donated blood as per the guidelines of the University Clinic at the University Freiburg. PMBC were isolated from buffy coats by Ficoll-Paque according to manufactures protocol (VWR, Germany), and CD14⁺ monocytes were isolated using CD14 magnetically labeled micro beads as per the manufactures protocol (Miltenyi Biotec, Germany). Monocytes were seeded at a concentration of 1.5 × 10⁶ cell/cm² and cultured for 4 days in alpha-MEM (Invitrogen, Germany) basic medium (HEPES, sodium pyruvate, penicillin–streptomycin-glutamine, 10% FCS) supplemented with 25 ng/mL rhM-CSF (RnD Systems, Germany) after which the media was supplemented with 50 ng/mL rhRANK-L (RnD Systems, Germany). Media was changed every 4 days, osteoclast formation was observed after 4 weeks of culture. Osteoclasts were stained with tartrate-resistant acid phosphatase (TRAP) staining kit (Sigma, Germany) according to manufactures protocol. The nuclei were stained with DAPI.