Cd14 clone m5e2

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from healthy donors (Oklahoma Blood Institute). Monocytes were enriched from PBMCs using magnetic bead separation (Miltenyi Biotech). Purity was >90% by flow cytometry and viability was >99% by trypan blue exclusion post enrichment. Monocytes were stimulated with 10 ng/ml recombinant hIL-10 (Peprotech) or recombinant vIL-10 (R&D systems) for indicated times. STAT3 phosphorylation was detected by flow cytometry using antibodies directed against phospho-STAT3 Y705 (BD Biosciences). To inhibit signaling through IL-10R, monocytes were stimulated with hIL-10 or vIL-10 in the presence or absence of a neutralizing antibody to IL-10R1 (Clone: 37607, R&D systems), and STAT phosphorylation was measured as above. To differentiate monocytes into macrophages, cells were cultured with 50 ng/ml M-CSF (R&D systems). On day 6 cells were additionally stimulated with IFNγ (20 ng/ml, Peprotech), IL4 (20 ng/ml, Peprotech), hIL-10 (10 ng/ml), or vIL-10 (10 ng/ml) for 24 h. Surface markers were stained using following antibodies: CD14 Clone M5E2, CD163 Clone GHI/61, CD32 Clone FLI8.26 (BD Biosciences), CD16 Clone 3G8, CD64 Clone 10.1, HLA-DR Clone LN3, CD86 Clone IT2.2 (BioLegend). Cells were acquired on BD LSR II or BD Celesta (BD Biosciences) and data were analyzed using FlowJo (TreeStar, v10).

Freshly isolated PBMCs were washed once in FACS buffer (PBS supplemented with 3% fetal bovine serum and 0.1% sodium azide) and stained for flow cytometry. Antibodies used; CD14 clone M5E2, HLA-DR clone G46-6, CD86 clone IT2.2, CD163 clone GHI/61, CD3 clone HIT3a, CD4 clone RPA-T4, CD8 HIT8a, CD25 clone 2A3, CD127-biotin clone HIL-7R-M21, CD56 clone B159, all from BD Biosciences. All analyzes were performed on a FACS Calibur and gated on viable PBMCs using 7AAD dead exclusion stain (BD Biosciences, San Jose, CA, USA).

Flow cytometry used to assess cell surface expression of host proteins was performed using a BD LSRFortessa (San Jose, CA, USA) instrument with FACS Diva software (San Jose, CA, USA), and all data were analyzed using FlowJo software version 10.7.1. (San Jose, CA, USA). HEK293 cells were stained with primary mouse anti-human monoclonal antibodies against α4β7 (clone ACT-1 [60 (link)]; NIH ARP), CD14 (clone M5E2; BD Bioscience, Sparks, MD, USA), CD162 (clone KPL-1; BD Bioscience), and CD81 (clone JS-81; BD Bioscience) for 30 min. After primary antibodies were removed by washing, staining with an R-phycoerythrin (PE) conjugated F(ab’)2-goat antimouse IgG secondary antibody which recognizes IgG heavy and light chains (Invitrogen, Carlsbad, CA, USA; Cat#A10543) was performed for 20 min. All antibodies were used at a concentration of 2 μg/mL for cellular staining.

Prior to treatments, THP-1 cell lines were diluted to 0.2x10⁶ cells/ml and differentiated with 100nM 1α,25-dihydroxy Vitamin D₃ (Cayman Chemical) for 72 hours. Differentiation was confirmed by measuring expression of CD11b and CD14 markers by flow cytometry (antibodies: CD11b, clone D12; CD14, clone M5E2, BD Pharmingen).

Cryopreserved PBMCs were thawed, washed, and stained with an antibody cocktail (1:100 dilution) of CD3 (clone SP34-2, BD Biosciences), CD4 (clone OKT4, Biolegend), CD8 (clone RPA-T8, BD Biosciences), CD14 (clone M5E2, BD Biosciences), CD20 (clone 2H7, Biolegend), IgM (MHM-88, Biolegend), IgG (clone G18-145, BD Biosciences) and fluorescently labeled biotinylated SIVmac239 SOSIP.664 Env at room temperature for 20 min in the dark. SIVmac239 Env trimer probes were labeled with two different fluorophores, from which SIVmac239 Env dual positive memory B cells (CD3⁻CD4⁻CD8⁻CD14⁻CD20⁺IgM⁻IgG⁺SIVmac239 SOSIP²⁺) were analyzed with BD FACSMelody or BD FACSFusion, and single-cell sorted, cultured, expanded in 384-well plates as described previously^{28 (link),29 (link)}. Briefly, sorted B cells were cultured with Iscove’s modified Dulbecco’s medium (IMDM) with GlutaMAX (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1× MycoZap Plus-PR (Lonza), 100 U/mL human IL-2 (Roche), 50 ng/mL human IL-21 (Invitrogen), 50 ng/mL human IL-4 (Miltenyi), 0.1 μg/mL anti-rhesus IgG (H + L) (BioRad), and irradiated 3T3msCD40L feeder cells. Flow cytometric data were subsequently analyzed using FlowJo (v10.7.1).

In order to detect which subset of human immune cells expresses DPP4, freshly isolated human PBMCs were incubated with a cocktail of fluorescence labelled monoclonal antibodies, i.e. anti-CD3 (clone SP34-2, BD Biosciences); CD56 (clone MEM188, Serotec); CD20 (clone B9E9, Beckman-Coulter); HLA-DR (clone L243, Biolegend); CD14 (clone M5E2, BD Biosciences); DPP4 (clone 222113, R&D Systems); and live/dead cell viability marker (Thermo Fisher Scientific). Appropriate Ig isotype control antibodies were used as negative controls for each staining. S1 protein of MERS-CoV that recognizes DPP4, used in a previous study^{25 (link)}, was applied to compare DPP4 expression in camel and human PBMC. Cells were analyzed on a Canto II flow cytometer (BD Biosciences) and using FlowJo® software (FlowJo), then subsequently presented as mean percentage of positive cells.