Anti cd14 clone mφp9

Whole blood was collected in Vacutainer tubes containing EDTA (Becton, Dickinson, USA) and 100μL aliquots were mixed in tubes with 2μL of undiluted monoclonal antibodies anti-CD14 (clone MΦP9) conjugated with peridinin chlorophyll protein complex (PerCP) (BD Pharmingen^™, USA). After erythrocyte lysis, cells were washed, permeabilized and incubated with monoclonal antibodies against MMP-2 (1A10) and MMP-9 (56129) (R&D Systems, USA) and IL-1β (AS10), TNF-α (6401.1111), TGF-β (9016) or IL-10 (JES3-9D7) (BD Pharmingen^™, USA) conjugated with phycoeritrin (PE) or fluorescein isothiocyanate (FITC). After incubation, cells were fixed, and phenotypic analyses were performed by flow cytometry using FACSCalibur cytometer (BD Biosciences, Breda, Netherlands). A total of 7x10⁴ neutrophils and monocytes (gated based on forward and side scatter plot) were collected and data were analyzed using FlowJo software. Neutrophils and monocytes analysis was performed using a dot plot of anti-CD14 (FL3)/SSC. The neutrophils were gated as SSC^HighCD14^low and monocytes were gated as SSC^lowCD14^High [30 (link)].

After thawing, PBMC were washed and immediately stained with the following panel: anti-CD3 (clone UCHT1; #IM2467, Beckman Coulter, Brea, CA, USA), anti-CD4 (clone SFCI12T4D11; #737660, Beckman Coulter), anti-CD8 (clone RPA-T8; #558207, BD Biosciences, Franklin Lakes, NJ, USA), anti-CD14 (cloneMφP9; #641394, BD Biosciences), anti-CD16 (clone3G8; #555406, BD Biosciences), anti-CD56 (clone N901; #A07788, Beckman Coulter), anti-CD11c (clone B-ly6; #561352, BD Biosciences), anti-CD19 (clone SJ25C1; #563036, BD Biosciences), anti-CD123 (clone 6H6; #45-1239-42, eBioscience, Affymetrix, Santa Clara, CA, USA), anti-HLA-DR (clone Immu-357; #IM3636, Beckman Coulter), and Zombie UV (#77474, Biolegend, San Diego, CA, USA). The samples were acquired on an BD Fortessa instrument equipped with the FACS Diva software. The analysis was performed with the FlowJo (FLOWJO, LLC, Ashland, OR, USA).

To examine PMN-MDSC, two-hundred thousand PBMC were stained with antibodies, comprising anti-CD66b (clone G10F5) conjugated to fluorescein isothiocyanate, anti-CD14 (clone MφP9) conjugated to allophycocyanin (both from BD Biosciences, San Jose, CA), CD11b (clone LM2) conjugated to phycoerythrin and anti-CD15 (clone W6D3) conjugated to peridinin chlorophyll protein complex (PerCP) (both from Biolegend, San Diego, CA). To provide insight into immunosuppressive function, we stained one million PBMC with anti-CD4 (clone RPA-T4) conjugated to PerCP to count CD4+ T cells in the same samples. Stained cells were acquired on a portable BD Accuri C6 flow cytometer (BD Biosciences).