Cd16 clone 3g8

Cell-surface markers were determined using the following fluorescence-labeled monoclonal antibodies specific to different lymphocyte subsets: T cells (CD3-FITC clone SP34-2, CD4-PerCP clone L200, and CD8-PE clone 3B5), B cells (CD20-APC clone L27), and NK cells (CD16 BV650 clone 3G8, along with CD3 antibody). All antibodies were purchased from BD Pharmingen, San Jose, CA, except CD8 antibody (Invitrogen). Monocytes and monocyte subsets were identified using CD14-AF700 clone M5E2 BioLegend, San Diego CA) and CD16 Clone 3G8 (BD). The methods of phenotype analysis of lymphocytes in peripheral blood were performed by surface staining of whole blood samples as described previously [30 (link), 32 (link)].

Ficoll-Paque was obtained from GE Healthcare (Uppsala, Sweden). Human Serum Albumin (HSA) was from Sanquin (Amsterdam, the Netherlands). HEPES-buffered RPMI 1640 was from Invitrogen (Carlsbad, CA, USA). Dihydrorhodamine 123 (DHR) was purchased from Sigma Aldrich (St. Louis, MO, USA) and dissolved in DMSO at a concentration of 3,33 mg/ml and stored at −20 °C. Platelet Activating Factor (PAF) and fMLF were purchased from Sigma Chemical Co (St. Louis, MO, USA). The VIM12 (CD11b, Mac-1, IgG1) was obtained from Caltag Invitrogen (Carlsbad, CA, USA). For flow cytometry staining we used the antibodies CD18 (clone L130); CD15 (clone MMA); CD32 (clone FLI8.26), CD35 (clone E11), CD44 (clone 515), CD63 (clone H5C6) and CD16 (clone 3G8) obtained from BD Pharmingen (San Diego, CA, USA). CD11b (clone 2LPM19c) was from DAKO (Copenhagen, Denmark), CD66b (clone 80H3) was from Cytognos (Salamanca, Spain), CXCR1 (clone 42705), CXCR2 (clone 48311) and CD45 (clone 2D1) were from R&D systems (Europe, UK) and CD64 (clone 10.1) was from AbD Serotec (Oxford, UK). CD29 (clone N29) was purchased from Millipore. All other chemicals were reagent grade.

To the whole blood culture tubes 3 mL were added phosphate-buffered saline to wash (PBS-W, 0.5% BSA, and 0.1% sodium azide), and centrifuged at 400 g for 10 min at 20°C. The supernatant was aspirated leaving a final volume of 2 mL. One hundred microliters of aliquots were mixed in tubes with 2 μL of undiluted monoclonal antibodies anti- CD14 (clone MΦP9) conjugated with peridinin chlorophyll protein complex (PerCP), CD16 (clone 3G8) conjugated with APC-Cy7 (BD Pharmingen™, USA), HLA-ABC (clone W6/32), HLA-DR (clone G46-6), TLR-2 (clone TL2.1), TLR-4 (clone HTA125), CD80 (clone 2D10) and CD86 (clone 2331), CD62L (DREG-56) and CD11b (ICRF44), conjugated with PE-Cy7, FITC, APC, or BV421. After erythrocyte lysis, the cells were washed, permeabilized and incubated with monoclonal antibodies against MMP-2 (clone 1A10), MMP-9 (clone 56129), NLRP3 (clone 768319), IL-1β (clone 8516), IL-10 (clone 127107), IL-12 (clone C11.5), IL-13 (clone JES10), IL-17 (clone BL168), IL-18 (clone 74801), IL-33 (clone 390412), IL-8 (clone E8N1), TNF (clone MAb11), TGF-β (clone TW4-9E7), TLR-9 (eB72-1665), and CASP1 (clone D-3) (Santa Cruz Biotechnologies, R&D Systems, BD Bioscience or Biolegend, USA) conjugated with distinct fluorescence. After incubation, the cells were fixed, and phenotypic analyses performed by flow cytometry using LSR Fortessa™ cytometer (BD Biosciences, USA).

PBMCs (10 000–50 000 cells) and isolated monocytes (5000–10 000 cells) were immediately stained for flow cytometry for a total of 20 min at 4°C. Due to inadequate sample amount, we were not able to perform all flow cytometric analyses on all patients. Antibodies used; CD14 clone M5E2 (1:10), HLA-DR clone G46-6 (1:50), CD80 clone L307.4 (1:15), CD86 clone IT2.2 (1:15), CD83 clone HB15e (1:15), CD33 clone WM53 (1:10), CD163 clone GHI/61 (1:15), CD16 clone 3G8 (1:20), CD3 clone HIT3a (1:25), CD4 clone RPA-T4 (1:25), CD8 HIT8a (1:25), CD25 clone 2A3 (1:10), CD127-biotin clone HIL-7R-M21 (1:10), CD56 clone B159 (1:10), all from BD Biosciences. Cells were analyzed using a FACSCalibur (BD Biosciences, San Jose, CA, USA). Analyzes were performed gated on PBMCs (≥2000 events per sample) and using 7AAD dead exclusion stain (BD Biosciences). Blood dendritic cell analyzes were performed using Blood DC enumeration kit according the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). For co-receptor expression, relative mean fluorescence intensity (MFI) was chosen to avoid any variability in antibody batches.

PBMCs were isolated from buffy coats as described earlier and pan-DCs (including myeloid and plasmacytoid DCs) were further enriched by negative selection using EasySep^TM human pan-DC pre-enrichment kit (STEMCELL Technologies) according to the manufacturer´s instructions. Cell purity of enriched live (fixable viability stain 780 ^– from BD Biosciences), lineage negative cells (CD14^– (clone M5E2, BD Biosciences), CD16^– (clone 3G8, BD Biosciences), CD3^– (clone UCHT1 BD Biosciences), CD20^– (clone 2H7, Biolegend) or CD19^– (clone HIB19, Biolegend), CD56^– (clone MEM-188, Biolegend), but HLA DR⁺ (clone L243, Biolegend)) was confirmed by flow cytometry. Isolated DCs were seeded at 1 × 10⁶ cells/ml in 96-well plates with 200 μl/well complete culture medium. Following 1–2 h rest, DCs were primed with 10% L. reuteri-CFS or were kept in RPMI as the control. Following 24 h incubation, cells were collected and washed twice with warm PBS and fresh medium with RA (1,15 μg/ml) or without RA was added. On day 4, the medium was replaced and on day 6 cells were re-activated with 10 μg/ml of Pam3SCK4 for 24 h. Supernatants were collected following 24 h priming with the first stimuli on day 1 and following 24 h stimulation with the second stimulus on day 7. Supernatants were stored at −20°C until further analysis.