Anti cd14 fitc

Cell surface and intracellular cytokine staining were performed as previously described (29 (link)). Monocyte phenotypes were analyzed by staining with anti-CD14-FITC (eBioscience), anti-CD16-PE (eBioscience), and anti-HLA-DR-eFluor® 450 (eBioscience) antibodies. For the intracellular staining of Foxp3⁺Helios⁺ Treg cells, we initially stained the cells for surface markers using the following antibodies: anti-CD4-PerCP-cyanine 5.5 (eBioscience), anti-human CD45RO-APC-eFluor® 780 (eBioscience), anti-human CD45RA-eFluor® 450 (eBioscience), and anti-human CD25-PE-Cy7 (eBioscience Inc., San Diego, CA). Cells were then fixed and permeabilized with the Foxp3 transcription factor staining buffer set, according to the manufacturer's instructions (eBioscience Inc., San Diego, CA), and the cells were then incubated with anti-Foxp3-PE (eBioscience) and anti-Helios APC (eBioscience Inc. San Diego CA) antibodies.

Cell surface and intracellular cytokine staining were performed as previously described [20 (link)]. Monocyte phenotypes and Foxp3⁺Helios⁺ Treg cells were stained with the following antibodies: anti-CD14-FITC (eBioscience), anti-CD16-PE (eBioscience), anti-HLA-DR-eFluor® 450 (eBioscience), anti-human CD279 (PD-1)-PerCP-eFluor® 710 (eBioscience); anti-CD4-PE-Cy7 (eBioscience), anti-xhuman CD45RO-APC-eFluor® 780 (eBioscience), anti-human CD45RA-eFluor® 450 (eBioscience), and anti-human CD25-PE-Cy7 (eBioscience) anti-Foxp3-PE (eBioscience), and anti-Helios APC (eBioscience Inc. San Diego CA).

The samples were submitted to the treatments described above and analyzed for the expression of the cell surface markers CD11b, CD14, C5aR1, C3aR, TLR2 and TLR4 on the surface of leukocytes labeled with specific antibodies for the monocyte (CD33) and granulocyte (CD66b) populations. After the blood treatment, red blood cells were lysed with BD FACS Lysing Solution buffer (BD Biosciences, California, USA). Subsequently, the cells were centrifuged at 405 g at 4°C for 10 minutes, resuspended and marked with monoclonal antibodies from BD Biosciences (California, USA) or eBioscience (California, USA), which were diluted in a 1:5 ratio with anti-CD11b PE (VIM12 clone), anti-CD14 FITC (clones 61D3 and TüK4) and anti-CD33 APC; in a 1:10 ratio with anti-C5aR FITC (clone 8D6), anti-C3aR PE (clone 17), anti-TLR2 PE (clone TL2.1) and anti-TLR4 PE (clone HTA125); and in a 1:20 ratio with CD3 APC-Cy7, CD19 PE-Cy7 and CD66b Alexa647. Monoclonal mouse IgG1k PE and IgG2ak FITC mice were also used as isotypic controls. After 30 minutes of incubation, 275 μL of FACS buffer was added, and the cells were analyzed in a FACSCanto II flow cytometer (BD Biosciences, California, USA) using SOFTWARE BD FACSDiVa, version 4.1 (BD Bioscience, California, USA). The results were expressed as median fluorescence intensity (MFI), determined from the acquisition of 20,000 events.

For surface staining of the stimulated cells, anti-CD4-APC (BD Biosciences), anti-CD3-PERCP, anti-CD8-FITC, anti-CD14-FITC (eBioscience, San Diego, CA, USA) were used. Fc receptor binding inhibitor antibody(eBioscience) was used to inhibit the non-specific Fc-gamma receptor (FcgammaR)-mediated binding of mouse monoclonal antibodies. Fix Viability Dye (eBioscience) was used to stain and exclude dead cells from the analyses. For intracellular staining, cells were restimulated on 50 ng/mL phorbol 12-myristate 13-acetate (Sigma-Aldrich) and 250 ng/mL ionomycin (Sigma-Aldrich) in the presence of 10 µg/mL of brefeldin A (Sigma-Aldrich) for the last 5 h of culture. Cells were fixed and permeabilized using the Cytofix/Cytoperm Fixation and Permeabilization Kit (BD Biosciences), labeled with anti-IL-17-PE (eBiosciences), and analyzed on the Gallios system using the Kaluza software (Beckman Coulter, CA, USA).

200 µL hirudin-anticoagulated whole blood was incubated at 37 °C with 15 mg MS@D-HMP in 1.5 mL PP tube. Negative control involved untreated whole blood, while positive control included whole blood treated with lipopolysaccharide (LPS, 10 ng/mL, Sigma-Aldrich). After 30 min, aliquots of the incubated whole blood were collected for assessment of the monocyte activation. 100 µL whole blood was stained with 5 µL anti-CD14-FITC (eBioscience™, #11-0149-42, clone 61D3) and 5 µL anti-CD11b-APC (eBioscience™, #17-0118-42, clone ICRF44). Following a 30-min dark incubation, 1.6 mL BD FACS™ Lysing solution was added to lyse red blood cells for 10 min and fix the remaining cells. Then, the samples were washed with 200 µL flow cytometry staining buffer (eBioscience™), and the volume was adjusted to 300 µL. The degree of monocyte activation was acquired using a flow cytometer (BD FACSCelesta™) by gating monocyte-specific events based on anti-CD14-FITC. The mean fluorescence intensities of monocyte activation marker CD11b-APC signal of monocyte population were normalized to the intensity of LPS-activated monocytes. All experimental acquisition was performed using BD FACSDiva™ software. Data analysis used FlowJo 10.6.2 software (TreeStar Inc).

Cryopreserved peripheral blood mononuclear cells (PBMCs) were used, and cell viability was evaluated. Cryopreserved PBMCs were thawed in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA), washed with PBS containing 1% BSA, and then incubated at room temperature for 20 min with the cell viability fixable viability stain 510 (BD Biosciences, San Jose, CA, USA). The viability of the PBMCs in the study is above 90%, as is shown in Figure 1. Cell surface staining were performed as previously described (15 (link)). After stained with anti-CD14-FITC (eBioscience), anti-CD16-PE (eBioscience), anti-CX3CR1-PE-Cy7 (eBioscience Inc., San Diego, CA) and anti-CCR2-APC-Cy7 (Biolegend Inc., San Diego, CA), monocyte phenotypes were detected by flow cytometry using a BD FACSCanto™ II with Diva software (BD Biosciences, San Jose, CA, USA). Then, data were analyzed by using FlowJo 10.0.7 software (Tree Star Inc., Ashland, OR, USA).