Magnetic bead separation

Human blood was sourced from healthy donors (Addenbrooke’s Hospital, Cambridge) and age, sex-matched CGD patients (Royal Free Hospital, London). Human mDCs were generated from CD14⁺ monocytes isolated from PBMC by magnetic bead separation (Miltenyi) and were differentiated by culturing for 6 days in RPMI1640 (Lonza) supplemented 5% FCS (Biosera), 20 ng/ml GM-CSF (Life Technologies) and 4 ng/ml IL-4 (BD Biosciences) as described previously (23 (link), 31 (link), 32 (link)). Naïve human CD4⁺ T cells were isolated by magnetic bead separation (Miltenyi).

Umbilical cord blood was collected after informed consent was obtained according to the Declaration of Helsinki. The ethics committee of the University Medical Center Utrecht approved these collection protocols. The protocol number of the ethical committee within the UMC Utrecht is TC-bio 15-345. CB mononuclear cells were isolated from human umbilical CB by density centrifugation over Ficoll-Paque solution (GE Healthcare Bio-Sciences AB, Chicago, IL, USA). CD34⁺ cells were isolated from fresh CB using magnetic bead separation (Miltenyi Biotec, Bergisch Gladbach, Germany) resulting in an 80–95% pure CD34⁺ population after running two columns, as determined with flow cytometry. CD14⁺ cells were isolated from fresh CB using magnetic bead separation (Miltenyi Biotec) resulting in a 95% pure CD14⁺ population.

IFN-γ ELISPOT assays on Ugandan samples were performed as described previously.^{44 (link)} Briefly, CD8⁺ T cells and APC were negatively selected from PBMC by one of two methods. From 2004 to 2008, cryopreserved PBMC were thawed and then depleted of CD4⁺ and CD56⁺ cells by magnetic bead separation (Miltenyi Biotec, Auburn, CA, USA). We determined empirically a priori that CD56+ cell depletion reduced the background in the assay. From 2009 to 2012, fresh whole blood was depleted of CD4⁺ cells using RosetteSep technology from StemCell Technologies. Using RosetteSep, we determined empirically a priori that CD56+ depletion was not necessary to eliminate background in the assay. CD4-depleted PBMC (250,000 cells per well) were then used as the source of responding CD8⁺ T cells and APC in an IFN-γ ELISPOT assay, using overlapping synthetic peptide pools consisting of 15-mers overlapping by 11 aa (final concentration 5 μg/ml) as a source of antigen. The ELISPOT assay was performed once on each donor. All determinations were performed in duplicate. Positive responses were defined as those that are 2SD above the media control, and greater than 10 SFU, which is a similar but more conservative rule than we have utilized previously in cohorts of young Ugandan children.^{44 (link)}

PBMCs were obtained from patients with HBV-ACLF who did not receive G-CSF therapy. CD14⁺ monocytes were further isolated using magnetic bead separation (Miltenyi, Bergisch Gladbach, Germany). The purity of monocyte separation was examined using flow cytometry, and CD14⁺ monocytes with a purity >95% were used in vitro experiments. Isolated monocytes were incubated in complete media with 20 ng/mL G-CSF or an equivalent volume of PBS (control) for 24 h for surface staining. For intracellular staining, the isolated CD14⁺ monocytes were stimulated with G-CSF (20 ng/ml) for 18 h. After 18 h incubation, LPS (100 ng/ml) and protein transport inhibitor (1μL/mL, BD GolgiPlug) was added and continue to incubation for 6 h, followed by staining with surface markers, and fixation, permeabilization, and staining with the corresponding fluorescent intracellular antibodies. After all the above steps, the monocytes were harvested and analyzed for their surface phenotype and intracellular cytokine levels using flow cytometry. Staining was performed as previously described.

Magnetic bead separation (130–049-201, Miltenyi Biotec, Germany) isolated mice splenic CD4+ T cells. The asthma model transfected BECs were pretreated with 10 nM of DHT [41 (link)], 1 nM of E2 [42 (link)], and 10 : 1 nM of DHT:E2 (see subsection 4.2.3) in severe asthma + DHT, severe asthma +E2, and severe asthma +DHT/E2 groups, respectively, for 24 h, cocultured with CD4+ T cells at a ratio of 10 : 1 (TCs: BECs) for 24 h in complete RPMI 1640 culture medium (Gibco, Australia) supplemented with 10% FBS, 1% penicillin and streptomycin, soluble anti-CD28 (1.0 μg/ml, eBioscience), soluble anti-cd3e (0.5 μg/ml, eBioscience), and IL-2 (20 ng/ml, eBioscience). To analyze T cell subsets, cells were collected after 24 h, and flow cytometry determined the concentration of IL-4 and IL-17A to obtain the ratio of Th2 to Th17 cells. BECs MBD2 was extracted by western blotting.

Daudi (ATCC® CCL-213™) B cells (40,000 per well) in RPMI-1640 with 10% ultra-low IgG were added to 96-well plates. Serial dilutions of indicated antibodies were added. CD56 + CD16 + NK cells were obtained from PBMC using magnetic bead separation (Miltenyi Biotech) and were added to plates (200,000 per well) resulting in an effector to target (E:T) ratio of 5:1. Plates were incubated for 4 h at 37 °C. After incubation, supernatant was transferred to 96-well plates and CytoTox® substrate mix added to each well (Promega; Madison, WI). Plates were incubated in the dark for 30 min then read at OD = 492 nm using SpectraMax® M3 spectrophotometer. Percent cytotoxicity of target cells was reported from triplicate measures and at least three donors as % cytotoxicity = (effector (spontaneous)—target (spontaneous))/(target (maximal)—target (spontaneous))*100, where medium background was subtracted from all values.