Cd14 bv421

CD14+ cells were cultured from haematopoietic cell precursors (CD34+) using a previously published protocol with minor modifications [35 (link)]. The culture cocktail contained X-VIVO10 (Lonza), Albumex (Seqirus) 0.05%, SCF (Peprotech) 200 ng/ml, GM-CSF (Peprotech) 0.03ug/ml, M-CSF (premium grade; Miltenyi Biotec) 5000U/ml, IL-6 (Peprotech) 10 ng/ml, FLT3 ligand (Peprotech) 50 ng/ml and gentamicin (Sigma Aldrich) 50 µg/ml. Calcitriol (1,25(OH)₂vitamin D₃; BioGems) was added at a physiological concentration of 0.1 nM. Cells were incubated at 37 °C with 5% CO₂ for 1 week before replating at a density of 1 × 10⁵ cells/ml of media. Media was then changed every third day by demi-depletion. Cells were harvested on day 21.
Harvested cells were centrifuged at 300×g for 5 min before the supernatant was removed. The cells were resuspended in 1 ml of chilled PBS, stained with 1 µl of LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies) and incubated on ice for 30 min. The cells were then washed and stained with the following antibody cocktail: CD45 BUV395 (BD Horizon), CD14 BV421 (BD), CD16 BV650 (BD), HLA-DR FITC (BD Pharmingen), CD34 PE (BD Pharmingen), CD11b APC (BD). FACS sorting was carried out on a BD Influx and CD14+ cells subjected to two washes with DPBS at 1000×g before storage at − 80 °C.

Lymphocyte surface phenotypes were evaluated by flow cytometry on cryopreserved PBMCs: CD4-APC-H7, CD8-PErCP-Cy5.5, CD38-PCY-7, HLA-DR-BV421, CD45RA-APC-H7, CCR7- PECy5, LIVE/DEAD-V500, Granzyme B-PE, Perforin-FITC, CD16-APC, CD14-BV421, CD56-PE (BD Biosciences, San Jose, CA, USA) and CD19-PercPVio700, CD80-APC (Miltenyi Biotec, Bergisch Gladbach, Germany). Combinations used were: CD4/CD8/CD38/HLA-DR (T-cell activation), CD14/CD16 (monocyte), CD16/CD56/CD3 (NK cells), CD11c/CD3 (DC), CD3/CD19/CD80 (B-cell activation), CD4/CD8/CD45RA/CCR7 (T-cell maturation). T-cell subsets were defined as naïve CCR (C-C chemokine receptor)7+CD45RA+, central memory (CM) CCR7+CD45RA−, effector memory (EM) CCR7−CD45RA−, terminally differentiated (TD) CCR7−CD45RA+. T follicular helper (Tfh) CD4+CxCR5+CD45RA+, T helper 17-like (Th17) CD4+CCR6+CD161+, T regulatory-like (Treg) CD4+CD127-CD25+. Briefly, 1 × 10⁶ PBMCs were stained with the appropriate antibodies for 20 min at 4°C in the dark and then washed and acquired using FACSVerse™ cytometer (BD Biosciences).

Blood samples were obtained under ketamine (7–10 mg/kg, IM) or telazol (3–6 mg/kg, IM) following an overnight fast. Procedures were performed at baseline and repeated following cycles three and six of PRF. Serum and plasma were separated by centrifugation, frozen, and stored at −80 °C until analyzed. Serum and whole blood were sent to Antech Diagnostics (Irvine, CA, USA) to assess blood chemistry and complete blood count values and to Metabolon (Durham, NC, USA) for LC–MS metabolome profiling. Hemoglobin A1C assays were performed using the Siemens DVA Vantage® analyzer to obtain a quantitative measure of the percent concentration of HbA1C in blood. All physiological measurements were collected within 1 h of sedation.
A 4 mL sample of whole blood was kept in an EDTA tube on ice for flow cytometry. Briefly, blood was treated with ACK to remove RBCs, and stained with conjugated monoclonal Abs (CD20-APC.Cy7, Biolegend clone 2H7; CD3-PE.Cy7, BD clone sp34-2; HLA-DR-ECD, BD clone immu-357; CD14-BV421, BD clone M5E2; CD8-APC, Biolegend clone SK1; CD4-FITC, BD clone L200; CD16-PE.Cy5.5, Biolegend clone 3G8; CD1c-PE, Militenyi Biotech clone AD5-8E7; CD123-PerCP.Cy5.5, BD clone 9F5) and Aqua-live/dead-BV510 (Thermofisher Scientific, L34597) for 30 min on ice, and measured on a BD FACSAria^TM Fusion. Analysis was performed with FlowJo (BD, NJ, USA).

IL-6 and Oncostatin M (OSM) induced STAT3 phosphorylation was assessed in the human erythroleukemia TF-1 cell line. Erythropoietin-induced STAT5 phosphorylation was assessed in the human UT-7 cell line. IL-2 and IL-15 induced STAT5 phosphorylation was assessed in activated human T-cells. Detection of phosphorylated STATs was accomplished with the SureFire pSTAT5 or pSTAT3 Assay kit (Perkin Elmer, Hopkinton, MA) per standard manufacturer’s protocol, with the exception of an overnight incubation following addition of donor beads before detection on the EnVision (Perkin Elmer, Hopkinton, MA). IFNγ induced STAT1 phosphorylation was assessed in the CD14+ monocyte population in human PBMC by flow cytometry. CD14 BV421 was purchased from BD Biosciences (San Jose, CA). STAT1 PE (pY705) was purchased from ThermoFisher Scientific (Waltham, MA). IL-4 and IL-13 induced STAT6 phosphorylation and IL-31 induced STAT3 phosphorylation were assessed in adult human epithelial keratinocytes (HEKa, ThermoFisher Scientific, Waltham, MA) by flow cytometry. STAT6 PE (pY641) and STAT3 PE (Y705) were purchased from BD Biosciences (San Jose, CA).