Cd14 hcd14

Blood was recovered in Vacutainer glass collection tubes with heparin (BD Biosciences). PBMC were isolated by gradient centrifugation with Histopaque-1077. Plasma was recovered for cytokine quantification by cytokine bead array (BD Biosciences) and resistin ELISA (Peprotech). Cell aliquots were frozen in liquid nitrogen. Following blood draw, PBMC isolation was performed within 24 hours through density gradient centrifugation, and cells were stored immediately in liquid nitrogen. Flow cytometry characterization of PBMC involved incubation with Human TruStain FcX™(Biolegend) and staining with primary antibodies: CD14(HCD14, Biolegend), CD16(3G8, Biolegend), CD66b(G10F5, eBioscience), CD3(OKT3, eBioScience). Samples were acquired on a BD LSRII and analyzed on FlowJo (v10).

Antibodies used for flow cytometry and immunofluorescent microscopy included those against human CD1b (SN13, Biolegend), Siglec-7 (QA79, eBioscience), CD14 (HCD14, Biolegend), CD56 (HCD56, Biolegend), CD34 (HM34, Biolegend), CD3 (UCHT1, BD Biosciences), CD19 (HIB19, Biolegend), HLA-DR (L243, Biolegend), CD11c (3.9, Biolegend), CD123 (6H6, Biolegend), EEA-1 (ab2900, Abcam) and LAMP-1 (ab24170, Abcam). For the blocking of LDN5 cell activation and liposome binding, anti-CD1b (BCD1b.3) (36 (link)) and anti-Siglec-7 (S7.7, Biolegend) were used, respectively. C80 GMM was isolated as described (36 (link)). Ficoll Plaque plus (GE Healthcare) was used for the density gradient centrifugation to separate peripheral blood mononuclear cells. Human IFNγ ELISA kit (Biolegend) was used to measure human IFNγ.

Cells were thawed at 37 °C, washed with sterile PBS and incubated with biotinylated anti-human lineage antibodies directed against CD2 (clone RPA-2.10, BioLegend), CD3 (HIT3a, 300304, BioLegend), CD4 (RPA-T4, BioLegend), CD7 (124-1D1, eBioscience), CD8a (RPA-T8, BioLegend), CD10 (SN5c, eBioscience), CD11b (ICRF44, BioLegend), CD14 (HCD14, BioLegend), CD19 (HIB19, BioLegend), CD20 (2H7, eBioscience), CD56 (HCD56, BioLegend) and GPA (HIR2, BioLegend) and LIVE/DEAD Fixable Aqua Dead Cell Stain (L-34957, Life Technologies). For gating on hematopoietic progenitor cells, this was followed by secondary staining with anti-human CD34–APC (8G12, BD Biosciences), anti-CD38–PE/Cy7 (HIT2, BioLegend), anti-CD90–FITC (5E10, BD Biosciences), anti-CD45RA-PB (MEM-56, Thermo Fisher Scientific), anti-CD123–PE (7G3, BD Biosciences) and streptavidin-APC/Cy7 (BioLegend). Intracellular staining with anti-human Ki-67–BV605 antibody (BioLegend) was performed using the BD Cytofix/Cytoperm Fixation/Permeabilization kit. Samples were run on an LSR II flow cytometer (BD Biosciences), and recorded events were analyzed with FlowJo 10 software (BD). Individual fluorescence-minus-one controls were used to determine gating. HSCs were identified as lineage⁻CD34⁺CD38⁻CD45RA^loCD90⁺ cells, CMPs as lineage⁻CD34⁺CD38^intCD45 RA⁻CD123^int cells and GMPs as lineage⁻CD34⁺CD38^intCD45RA⁺CD123^int cells.