Anti human cd14 magnetic beads

“Peripheral blood mononuclear cells were obtained from blood leukocyte preparations purchased from the New York Blood Center, by density gradient centrifugation with Lymphoprep (Stemcell Technology, Vancouver, BC, Canada) using a protocol approved by the Hospital for Special Surgery institutional review board”.^{48 (link)} “CD14⁺ monocytes were obtained from peripheral blood, using antihuman CD14 magnetic beads, as per the manufacturer’s protocol (Miltenyi Biotec, Auburn, CA, USA). Purity of monocytes was >97%, as verified by flow cytometric analysis. CD14 ⁺ cells were plated at a density of 1 × 10⁶ cells·mL⁻¹ and cultured with 20 ng·mL⁻¹ of M-CSF (Peprotech) in alpha modified essential medium (α-MEM) (Thermo Fisher Scientific) supplemented with 10% Hyclone fetal bovine serum (GE Healthcare) and 1% L-glutamine (200 mmol·L⁻¹, Thermo Fisher Scientific) for 1 day to obtain OCPs”.^{25 (link)} OCPs then were incubated with 20 ng·mL⁻¹ of M-CSF and 40 ng·mL⁻¹ of human soluble RANKL (Peprotech) to differentiate into osteoclasts. “Medium and cytokines were replenished every 3 days. When multinucleated cells were observed, cells were fixed and stained for TRAP using the Acid Phosphatase Leukocyte diagnostic kit (Sigma Aldrich) as recommended by the manufacturer. Multinucleated (>3 nuclei), TRAP-positive osteoclasts were counted in triplicate wells”.^{48 (link)}

Leukocyte apheresis cones were collected from healthy donors at the National Blood Service (Manchester, UK). PBMCs were separated by centrifugation using Ficoll-Paque (GE Healthcare, Amersham, UK). Monocytes were separated from PBMCs using anti-human-CD14 magnetic beads (Miltenyi Biotec, Cologne, Germany) according to the manufacturer’s instructions, using an LS MACS separation column. Monocyte purity was consistently over 95%.
Monocytes were cultured in StemXvivo serum-free DC base medium (Bio-techne, Minneapolis, MN, USA) containing 25 ng ml⁻¹ GM-CSF and 25 ng ml⁻¹ interleukin-4 (Biolegend, San Diego, CA) for 6 days at a concentration of 0.5 × 10⁶ cells per ml in 24-well tissue culture-treated plates. Half of the medium was removed on day 3 and replaced with fresh medium and cytokines. After 6 days of differentiation, cells were treated for 48 h with different combinations of compounds: 5 ng ml⁻¹ active TGFβ (Peprotech, Rocky Hill, NJ), 100 μg ml⁻¹ anti-TGFβ antibody (clone 1D11, West Lebanon, BioXcell, NH), 1 µM pan-RA receptor antagonist (LE540, a kind gift from Hiroyuki Kagechika, Tokyo Medical and Dental University, Tokyo, Japan), 100 nM RA (Sigma Aldrich, St Louis, MO, USA), 1 µg ml⁻¹ Pam3CSK4, 10 μg ml⁻¹ Poly(I:C), 1 µg ml⁻¹ flagellin, 5 μg ml⁻¹ Imiquimod, 5 μg ml⁻¹ ssRNA40, or 100 ng ml⁻¹ LPS (all from Invivogen, San Diego, CA).

Venous blood was collected in Sodium-Heparin vacutainers (Cat# 367874, BD). Plasma and blood cells were separated using Histopaque-1077 (Cat# 10771, Sigma) according to the manufacturer’s protocol. Briefly, whole blood was diluted 1:1 with sterile DPBS-2 mM EDTA (Sigma or Corning) and overlaid (30 mL) on to 10 mL of Histopaque-1077. Gradients were centrifuged at 500xg for 30 min. The peripheral blood mononuclear cell (PBMC) layer at the plasma-Histopaque interface was transferred to a new tube and washed twice with cold DBPS-EDTA. PBMCs were isolated and CD16+ cells depleted using anti-human CD16 magnetic beads (Cat# 130-045-701, Miltenyi) using manufacturer’s protocol. After CD16 depletion, CD14+ monocytes were purified using anti-human CD14 magnetic beads (Cat# 130-050-201, Miltenyi). Monocytes were plated in 24-well tissue culture treated plates at density 5x10⁵ per well in 1 mL of cRPMI supplemented with human recombinant M-CSF (50 ng/ml; Peprotech). Every two days the media was exchanges for fresh cRPMI containing M-CSF. At the day 7-8 the media was changed to cRPMI without M-CSF and stimulations were carried out as indicated. MoDMs were stimulated with LPS (100 ng/ml) and ATP (5 mM, 45 min).