Ficoll paque

CD14⁺ monocytes were isolated from blood of human donors (approved by the local ethical committee, 2007–2019, with written consent obtained from each donor) by centrifugation through Ficoll-Paque (Amersham, GE Healthcare, Little Chalfont, UK), and subsequently suspended in 0.5% bovine serum albumin (BSA) and 2 mM EDTA in PBS, and were purified by using BD IMag™ Anti-Human CD14 Magnetic Particles - DM (BD Biosciences, CA) according to the supplier's instructions (Moller et al., 2017 (link)). CD14⁺ cells were seeded at a density of 66,667 cells/cm² in T75 culture flasks (Greiner, InVitro, Fredensborg, Denmark) supplied with α minimum essential medium (αMEM; Invitrogen, Taastrup, Denmark) containing 10% fetal calf serum (FCS; Sigma-Aldrich, St Louis, MO) and 25 ng/ml human macrophage colony-stimulating factor (M-CSF; R&D Systems, Abingdon, UK) and cultured for 2 days at 37°C in 5% CO₂ in a humidified atmosphere (Soe and Delaisse, 2010 (link)). Floating cells were harvested by centrifugation (500 g for 5 min) and returned to the respective flasks in fresh αMEM with 10% FCS, 25 ng/ml human M-CSF and 25 ng/ml human RANKL (also known as TNFSF11). The cells were cultured for an additional 7 days with medium changed twice.

K562 cells were cultured in RPMI supplemented with 10% FBS as described previously32 (link). Mononuclear cells were isolated from umbilical cord blood by Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) density gradient centrifugation after obtaining the donor’s consent according to governmental regulations (“Guidelines for collection and use of human specimens for research”, Ministry of Health and Welfare, Taiwan) approved by the Institutional Review Board of the Taoyuan General Hospital, Taiwan. The isolation and culture methods for hHSC were described previously17 (link). Briefly, human CD34⁺ hHSC were isolated from mononuclear cells by magnetic micro-bead isolating methods using Direct Progenitor Isolation Beads (Miltenyi Biotech, Bergisch Gladbach, Germany) and MACS LS-columns (Miltenyi Biotech). 2.5 × 10⁵ hHSC cells were cultured in 5 ml of EDM (5 × 10⁴ cells/ml) in the presence of SCF (50 ng/ml) and EPO (6 IU/ml) for 6 days.

PBMC were isolated by differential centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech). CD4⁺ and CD14⁺ cells were isolated by positive immunomagnetic selection (MiltenyiBiotec), and were >95% CD4⁺ and CD14⁺, respectively, as measured by flow cytometry. CD4⁺ cells were cultured in 12-well plates at 2 ×10⁶ cells/well in RPMI 1640 containing 10% heat-inactivated human serum, with 2 × 10⁵ autologous CD14⁺ monocytes/well and γ-irradiated M. tb (10 µg/ml) at 37°C. After 4 days, CD4⁺CD25⁺ and CD4⁺CD25^- cells were isolated, using the Treg isolation kit (MiltenyiBiotec), as described [11 (link)]. Eighty five-90% of isolated CD4⁺CD25⁺ cells expressed Foxp3, and <1% of CD4⁺CD25^- cells were Foxp3⁺, as determined by intracellular staining.

Spleens were obtained from post-mortal kidney donors (Erasmus MC Hospital, Rotterdam) and anonymously used for research purposes as described in article 13 of The Netherlands law of organ donation (Wet op Orgaandonatie, WOD). All samples and data were analyzed anonymously. Spleens were mechanically disrupted and filtered through a 70-µm cell strainer (Greiner Bio-one, Alphen a/d Rijn, The Netherlands) to obtain a single-cell suspension. Mononuclear cells, isolated using Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient, were stored at −150°C until use. Upon thawing, quiescent B cells were isolated by negative selection using anti-CD43-magnetic beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) (23 (link)). Purity was determined by flow cytometry (FACS Canto II). Typically, cell suspensions consisted of >98% pure CD19⁺ B cells. B cells from spleens from 12 different donors were used it the experiments.