Ficoll paque density centrifugation

Mononuclear cells (MNCs) from two up to five CB collections were pooled and purified by Ficoll-Paque density centrifugation (GE Healthcare Life Sciences, Buckinghamshire, UK) followed by ammonium chloride red cell lysis. Density-separated CB MNCs were magnetically sorted for CD34 positivity via the EasySep Human CD34 Positive Selection kit (Stemcell Technologies) according to the manufacturer’s instructions; alternatively, they were sorted by fluorescent activating cell sorting (FACS). Foetal liver (FL) CD34⁺ were isolated from human foetal tissue ranging from 12 to 20 weeks post-conception (wpc). Tissue digestion was performed at 37 °C using an enzymatic solution (0.1 U/mL Collagenase A (Roche), 0.8 U/mL Dispase II (Roche) and 100 μg/ml DNase I (Roche) in RPMI, 2% FBS and 1% Penicillin/Streptomycin). Cells were pelleted and processed for ammonium chloride red cell lysis. FL MNCs were magnetically sorted for CD34 positivity.

In vivo labelling was performed as described previously. Mice were intravenously injected with 2.5μg PE/Pacific Blue-conjugated anti-CD45 or anti-CD19 in PBS. After 4min mice were euthanized and lungs perfused with 10ml cold PBS through the right ventricle. Lungs were removed and roughly dissected with scissors before digestion in 1mg/ml collagenase D (Roche) and 10μg/ml DNAseI in RPMI for 45min at 37°C. Tissue was homogenised through a 70μm mesh and for experiments assessing BRM, lymphocytes were enriched by Ficoll-Paque density centrifugation (GE Healthcare). Cell suspensions were incubated with FC block in FACS buffer (2% FBS, 0.1% Sodium Azide, 1mM EDTA in PBS) for 15min and then stained in FACS buffer using predetermined optimal antibody concentrations for 30min. Cells were then washed and labelled with secondary labelling agents for 20min. For intracellular staining of antibody isotypes, Cytofix/Cytoperm Staining Buffer Kit (BD Biosciences) was used as per manufacturer’s instructions. Data acquisition was performed using a BD Fortessa X20 (BD Biosciences) and analysed using FlowJo v10.8 (Tree Star Inc.). For BRM cell phenotypic characterisation (Figures 1B and 1C), data acquisition was performed on a Cytek Aurora (Cytek Bioscienes). For cell sorting, samples were prepared as above and sorted using a FACSAria III (BD Biosciences).

Venous whole blood (approximately 18 ml) was collected from study participants, with two additional whole blood spots for diagnosis of Plasmodium infection. Peripheral blood mononuclear cells (PBMCs) were purified by Ficoll-Paque density centrifugation according to manufacturer’s instruction (GE Healthcare, Uppsala, Sweden). CD14+ monocytes were isolated with EasySep CD14 positive selection kit according to manufacturer’s instructions (Stem Cell Technologies, Grenoble, France). CD14+ monocytes and the remaining CD14- cells (lymphocytes) were stored in RNAprotect Cell Reagent (Qiagen, Hilden, Germany) at −80°C (Materials and Methods - Supplement Figure 1). Plasma was obtained following centrifugation of venous whole blood, and stored at −80°C.

Human whole peripheral blood (PB) was obtained from healthy adult volunteers following written informed consent. PBMCs were isolated by Ficoll-Paque density centrifugation (GE Healthcare, Freiburg, Germany). Primary NK cells were purified using the EasySep Human NK Cell Isolation Kit (Stemcell Technologies, Vancouver, YVR, Canada) according to the manufacturer’s protocol. The cells were then co-cultured with target cells to assess their efficacy according to methods described below.

Blood was obtained from specific pathogen free (SPF) Swiss Large White pigs. The blood sampling was approved by the cantonal ethical committee for animal experiments, license #BE88/14. Peripheral blood mononuclear cells (PBMC) were isolated using ficoll-paque density centrifugation (1.077 g/L; GE Healthcare Life Sciences, Dübendorf, Switzerland). Monocytes were sorted as CD172a+ cells using monoclonal antibody (mAb), clone 74-22-15A (hybridoma kindly provided by Dr. A. Saalmüller, Veterinary University of Vienna, Austria) and magnetic cell sorting with LS columns and the MACS (magnetic cell sorting) system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Macrophages were generated as previously described [26 (link)]. Briefly, monocytes were cultured at 5 × 10⁵ cell/mL in Dulbecco’s modified Eagle’s medium (DMEM) containing Glutamax (ThermoFisher Scientific, Zug, Switzerland) supplemented with 10% of specific pathogen-free porcine serum (produced in-house), seeded in 24-well culture plates and incubated for three days at 39 °C and 5% CO₂.