Cd14 percp

Cryopreserved PBMCs from 21 Löfgren’s syndrome patients were thawn and resuspended in phosphate-buffered saline (PBS). The cells were stained with CCR5-PECy7 (Ebioscience, San Diego, CA, USA), CD14-PerCP (monoclonal Peridinin-Chlorophyll-Protein, PerCP-labelled antibody, Becton Dickinson, San Jose, CA, USA), and CD16 PE (Phycoerythrin labeled antibody, Becton Dickinson). Mouse IgG1 kappa Isotype Control PE-Cy7 (Ebioscience) was used as negative control. The cells were measured on a FACS-Calibur (BD Biosciences, San Jose, CA, USA) and data analysis was performed using FlowJo software (v10.7, Ashland, OR, USA). Gating strategies for differentiation between classical, intermediate, and non-classical monocytes were performed as described before [17 (link)]. Monocytes were first gated according to their size and granularity characteristics in a FSC-SSC plot and then for CD14 expression. The percentage of CCR5 positive cells as well as the CCR5 median fluorescence intensity (MFI) expression levels were determined on CD14⁺ monocytes.

PBMCs were isolated from whole blood collected from family members and healthy donors by ficoll-histopaque gradient centrifugation. For phenotypic staining, the following monoclonal antibodies were used: CD3-APC-H7, CD4-PerCP-Cy5.5, CD38-PerCP-Cy5.5, CD10-PECF594, CD21-APC, IgG PeCy7, CD14-PerCP, CD123-PE, CD56-PeCy7, CD11c-APC, CD16-APC-H7 (BD Biosciences, San Diego, CA, USA), CD8-APC-EF780, CD27-APC-EF780 (eBioscience, San Diego, CA, USA), CD19-BV650, CD24-BV605 (Biolegend, San Diego, CA, USA) and IgA-PE (Miltenyi Biotech, Bergisch Gladbach, Germany). Naïve B cells were enriched by negative selection using B-cell isolation kit (Stemcell, Vancouver, BC, Canada). Naïve B-cell purity was verified by flow cytometry to 98% purity.

The antibodies against CD14-PerCP, CD16-PECy7, CD86- APC, CD61–FITC, CD62P-PE, unlabeled CD62P and FACS™-Lysing solution were obtained from BD Bioscience (San Jose, CA, USA). Thrombin receptor activator peptide (TRAP – 10 µM) was purchased from Sigma Aldrich (St Louis, MO) and abciximab [an antibody against glycoprotein IIb/IIIa inhibiting platelet aggregation (ReoPro®; 2 mg/ml)] from Lilly Benelux S.A/N.V.

Platelet-depleted blood (40 μl) was incubated with E. coli (10⁷/ml) in a rolling incubator at 37°C for 15 minutes. After adding 6.4 μl of the stop solution, the leukocytes were stained with antibodies: CD45-Pacific Orange (catalog no. MHCD4530, Invitrogen™), CD14-PerCP (catalog no. 340585, BD Biosciences), CD15-V450 (catalog no. 48-0158-42, Invitrogen™), CD11b-APC/Fire 750 (catalog no. 101262, BioLegend, San Diego, CA), and CD35-Alexa Fluor 647 (catalog no. 1981978, Invitrogen™) for 15 minutes at 4°C. After lysing the red blood cells with fixative-free lysis buffer (Invitrogen™, catalog no. HYL250), the solution was centrifuged at 250×g, 4°C for 5 minutes. After centrifugation, the supernatant was discarded and the pellet was resuspended in PBSA. The expression of the abovementioned markers was analysed with Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific). Granulocytes were gated as CD15+ population (Supplementary Figure 2), while monocytes were gated as CD15- and CD14+ population (Supplementary Figure 3). The activation of granulocytes and monocytes was evaluated by the expression levels of CD11b and CD35 on their surfaces. Flow data analysis was performed with FlowJo version 10.

CD14⁺ cells were purified from PBMC using auto-MACS Pro by positive-selection. Monocyte purity was measured by flow cytometry after staining with mouse-antihuman monoclonal antibodies against CD14-PerCP (BD Biosciences) and CD3-PacBlue (BD Biosciences). Isolated CD14⁺ monocytes (2 × 10⁵ cells/200 µl) were cultured in RPMI 1640 medium (Life Technologies), supplemented with 10% FBS and 1% P/S. Monocytes were cultured with MP, or MPγ at different ratios (1:10,000, 1:40,000, 1:80,000) in polypropylene tubes. After 24 h of incubation, monocytes were collected for PCR analysis or flow cytometry after staining with CD14-PacBlue, CD3-PerCP, CD16-FITC, PD-L1-PE and CD90-APC (all BD Biosciences).