Anti cd14 percp cy5

Antibodies (Abs) used for flow cytometry were anti-IFN-γ-PE-Cy7 (BD Pharmingen); anti-CD66b-FITC, anti-CD33-PE, anti-CD45-PE-Cy7, anti-CD14-PerCP-Cy5.5, anti-HLA-DR-Alexa Fluor 647 and CD8-PerCP-Cy5.5 (eBioscience); anti-perforin-PE, and anti-granzyme B-APC (Biolegend). Abs used for western blotting were anti-human arginase I (R&D Systems) and horseradish peroxidase (HRP)-conjugated secondary ab (Zhongshan Biotechnology). Abs used for IL-6/IL-8 blockade studies were: anti-human IL-6 (Biolegend) and anti-human IL-8 (R&D Systems). The chemical arginase I inhibitor nor-NOHA (Santa Cruz) was also used. Purified anti-CD3 and anti-CD28 Abs (Biolegend) and recombinant human IL-2 (PeproTech) were also used for T cell studies.
Recombinant human IL-6, IL-8, and arginase I (Biolegend) were used for CD45⁺CD33^lowCD11b^dim activation studies. Drug inhibitors studies involved the STAT3 inhibitor FLLL32 (Medco Biosciences), PI3K inhibitor Wortmannin, and MAPK inhibitor SB203580 (Calbiochem).

For expression analysis of macrophage marker surface proteins and TLR2, undifferentiated and differentiated as well as unstimulated and/or pam3CSK4-stimulated cells were resuspended in Flow Cytometry Staining Buffer (eBioscience, San Diego, CA, USA). To block Fc receptors and prevent nonspecific binding of Fc fragments, cells were incubated with Human Fc Receptor Binding Inhibitor Purified (eBioscience). 1 × 10⁶ cells were stained with anti-TLR2-FITC (mouse monoclonal, FAB2616F, R&D); anti-CD11b-FITC (mouse monoclonal, 11-0113, eBioscience); anti-CD11b-APC (mouse monoclonal, 17-0118, eBioscience); anti-CD14-PerCP-Cy5.5 (mouse monoclonal, 45-0149, eBioscience) or with appropriate isotype controls. Flow cytometric analysis was performed on LSRFortessa (BD Biosciences, San Jose, CA, USA) instrument and analyzed using FlowJo software (Tree Star Inc.). Since these experiments were performed for quality control reasons, to demonstrate the proper molecular characteristics of our cellular models, we do not include these results as figures in the results section. Representative flow cytometry histograms may be found in supplementary material as Supplementary Figure 1 (differentiation markers) and Supplementary Figure 2 (TLR2 expression).

Bone marrow derived-macrophages were isolated according to previously published methods(60) and differentiated in the presence of L929 conditioned media for 8 days as per standard protocols. After 8 days the cells were incubated with 4 mg/ml lidocaine (Sigma) for 15 min at 4°C and gently lifted using a cell lifter. Cells were then centrifuged, counted and re-suspended in medium at a concentration appropriate for measurement of cytokine production, bacterial uptake, flow cytometry or bacterial killing assays. Macrophage maturation was assessed by flow cytometry using APC-conjugated anti-F4/80, PE-conjugated anti-Ly6G or -CCR2, FITC-conjugated Ly6C, eFluor 450-conjugated CD45 and PE-Cy7-conjugated CD11b, or corresponding isotype controls. Pattern recognition receptor (PRR) expression was measured using anti-TLR4-FITC, anti-TLR2-PE-Cy7 and anti-CD14-PerCpCy5.5 (eBioscience), as well as anti-MARCO-PE (RND systems). To visualize S. pneumoniae uptake by macrophages, TRITC labeled bacteria were incubated with bone marrow derived macrophages for 2h at an MOI of 200. Cells were fixed and stained using an anti-beta actin antibody (Cell Signaling). Images were acquired at 40X magnification using an inverted Zeiss LSM510 laser confocal microscope.