japan recombinant human cd14 protein rcd14 383 cd r d systems minneapolis  (R&D Systems)

Journal: bioRxiv

Article Title: Mesenchymal Stromal Cells Immunosuppress Osteoarthritis Synovial Fluid Tolerized Monocytes via IL-6

doi: 10.1101/2025.08.14.669875

Figure Lengend Snippet: (A) Frequency of classical (CD14 + CD16 neg ) and intermediate (CD14 + CD16 + ) peripheral CD14 + monocyte subsets with and without OA synovial fluid (SF). (B) Percent intermediate (CD14 + CD16 + ) monocytes in CD45 + cells with indicated treatments. (C, D) Mean fluorescence intensity (MFI) of CD163, CD206, HLA-DR, and CD86 surface markers, and HLA-DR/CD163 MFI ratio on peripheral CD14⁺ monocytes from late-OA patients and healthy donors, with or without late-stage OA SF, following lipopolysaccharides (LPS, 2.5 ng/mL) stimulation. (E-I) MFI expression of CD163, CD206, HLA-DR, and CD86 cell surface markers and HLA-DR/CD163 MFI expression ratio on healthy peripheral CD14 + monocytes at the indicated conditions. (J) Tumor necrosis factor (TNF) soluble factor production by peripheral CD14 + monocytes in indicated treatments, with LPS (2.5 ng/mL) stimulation. Peripheral CD14 + monocytes were cultured for 72h with indicated treatments in (A-I) for flow cytometry assay and 48 hours in (J) for TNF assay; IL-6 recombinant (5 ng/mL); IL-6 neutralizing antibody (Ab; 2.5 µg/mL); 30% pooled mid- or late-OA SF. Values are shown as mean ± SD; N=3 biological replicates; n ≥ 3 technical replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was used. Letters indicate significant differences (p< 0.05) between groups; asterisks indicate significance (p<0.05) relative to untreated controls.

Article Snippet: The phosphorylation of protein kinases in CD14+ monocytes treated with late-stage OA SF for 1 hour was assessed using the Human Phospho-Kinase Array Kit (R&D Systems; ARY003C), according to the manufacturer’s instructions.

Techniques: Fluorescence, Expressing, Cell Culture, Flow Cytometry, Recombinant

Journal: bioRxiv

Article Title: Mesenchymal Stromal Cells Immunosuppress Osteoarthritis Synovial Fluid Tolerized Monocytes via IL-6

doi: 10.1101/2025.08.14.669875

Figure Lengend Snippet: (A) Schematic representation of dual IL-6 signaling in cells such as monocytes/macrophages (MΦs) expressing IL-6 receptor (IL-6R): i) trans-signaling through glycoprotein (gp)130 and soluble IL-6R (sIL-6R shed by a disintegrin and metalloproteinase 17 (ADAM 17)); ii) classical signaling through membrane-bound (m) IL-6R and gp130. (B) Median and interquartile range of sIL-6R concentration in synovial fluid (SF) samples from not-OA (N=7); mid-OA (N=24); and late-OA (N=43). (C-F) Mean fluorescence intensity (MFI) expression of CD163, CD206, HLA-DR and CD86 cell surface markers in the presence of late-OA SF and physiological levels of IL-6 (0.5 ng/mL) or/and sIL-6R (10 ng/mL), soluble(s) gp130 (10 ng/mL). (G) mIL-6R expression on CD14 + monocytes at the indicated treatments. (H) IL-6: sIL-6R complex levels in not-OA SF (N=7); mid-OA SF (N=24); late-OA SF (N=72), psoriatic arthritis (PsA) SF (N=2) and rheumatoid arthritis (RA) SF (N=1). Peripheral CD14 + monocytes were cultured for 72h with 30% pooled mid- or late-OA SFs, IL-6 neutralizing antibody (Ab; 2.5 µg/mL), and indicated treatments. MFI expression of monocyte cell surface markers is shown as mean ± SD; N=3 biological replicates; n=3 technical replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was used. Letters indicate significant differences (p< 0.05) between groups; asterisks indicate significance (p<0.05) relative to untreated controls.

Article Snippet: The phosphorylation of protein kinases in CD14+ monocytes treated with late-stage OA SF for 1 hour was assessed using the Human Phospho-Kinase Array Kit (R&D Systems; ARY003C), according to the manufacturer’s instructions.

Techniques: Expressing, Membrane, Concentration Assay, Fluorescence, Cell Culture

Journal: bioRxiv

Article Title: Mesenchymal Stromal Cells Immunosuppress Osteoarthritis Synovial Fluid Tolerized Monocytes via IL-6

doi: 10.1101/2025.08.14.669875

Figure Lengend Snippet: (A) An experimental timeline for CD14⁺ monocytes indicating treatment conditions at each time point (arrows) for both short- and long-term exposure periods. (B) Representative western blot images from CD14 + monocytes at short treatment exposures. (C) Representative western blot images from CD14 + monocytes at long treatment exposures. (D-F) Bar graphs illustrating phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), phosphorylated-c-Jun terminal kinase (p-JNK), and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) levels (normalized to total glyceraldehyde-3-phosphate dehydrogenase; GAPDH) in short and long treatment exposures. (G) Suppressor of cytokine signaling 3 ( SOCS3 ) expression in CD14 + monocytes with indicated treatments. (H) Chemokine (C-C motif) ligand 2 ( CCL2 ) expression in CD14 + monocytes with indicated treatments. (I) Tumor necrosis factor ( TNF ) expression by CD14 + monocytes in indicated treatments and lipopolysaccharides (2.5 ng/mL) stimulation. Peripheral CD14 + monocytes were cultured for 48h (G-I); IL-6 recombinant (5 ng/mL); S100A8/9 recombinant (1 µg/mL); IL-6 neutralizing antibody (Ab; 2.5 µg/mL); 30% pooled late-OA synovial fluid (SF); TAK242 (1 µM/mL). Values are shown as mean ± SD; N=3 biological replicates; n≤3 technical replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was used. Letters indicate significant differences (p< 0.05) between groups; asterisks indicate significance (p<0.05) relative to untreated controls.

Article Snippet: The phosphorylation of protein kinases in CD14+ monocytes treated with late-stage OA SF for 1 hour was assessed using the Human Phospho-Kinase Array Kit (R&D Systems; ARY003C), according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Cell Culture, Recombinant

Journal: bioRxiv

Article Title: Mesenchymal Stromal Cells Immunosuppress Osteoarthritis Synovial Fluid Tolerized Monocytes via IL-6

doi: 10.1101/2025.08.14.669875

Figure Lengend Snippet: (A) Experimental timeline and schematic workflow for CD14 + monocytes directly co-cultured with MSC(M), 30% pooled mid- or late-OA synovial fluid (SF) or IL-6 neutralizing antibody (Ab; 2.5 µg/mL) for 72h. (B-F) Mean fluorescence intensity (MFI) expression of CD163, CD206, HLA-DR, CD86 cell surface markers and the ratio of HLA-DR/CD163 MFI expression on CD14 + monocytes directly co-cultured with MSC(M) and indicated treatments. Values are shown as mean ± SD; N=3 biological replicates; n≤3 technical replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was used. Letters indicate significant differences (p<0.05) between groups; asterisks indicate significance (p<0.05) relative to untreated controls.

Article Snippet: The phosphorylation of protein kinases in CD14+ monocytes treated with late-stage OA SF for 1 hour was assessed using the Human Phospho-Kinase Array Kit (R&D Systems; ARY003C), according to the manufacturer’s instructions.

Techniques: Cell Culture, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Mesenchymal Stromal Cells Immunosuppress Osteoarthritis Synovial Fluid Tolerized Monocytes via IL-6

doi: 10.1101/2025.08.14.669875

Figure Lengend Snippet: (A) Experimental timeline and schematic workflow for CD14 + monocytes treated with soluble factors from licensed MSC(M) CM in OA environment for the indicated times. (B) Representative western blot images. (C-E) Bar graphs illustrating phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), phosphorylated-c-Jun terminal kinase (p-JNK), and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) levels (normalized to total glyceraldehyde-3-phosphate dehydrogenase; GAPDH) after 24h of treatments. (F) Expression of the suppressor of cytokine signaling 3 ( SOCS3) gene in treated CD14 + monocytes. (G and H) JNK downstream chemokine (C-C motif) ligand 2 ( CCL2) and tumor necrosis factor ( TNF) genes expression in treated CD14 + monocytes. (I) TNF soluble factor production by peripheral CD14 + monocytes in indicated treatments and lipopolysaccharides (2.5 ng/mL) stimulation. (J) Phagocytosis of pHrodo Deep Red E. coli BioParticles by macrophages in indicated treatments for 72h. Peripheral CD14 + monocytes were treated with CMs of directly co-cultured monocytes with MSC(M), and mid- or late OA SF ± IL-6 neutralizing antibody (Ab, 2.5 µg/mL); 30% pooled mid- or late synovial fluid (SF). Gene expression in (F-H) and TNF soluble in (I) were assessed after 48h. Values are shown as mean ± SD; N=3 biological replicates; n ≥ 3 technical replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was used. Different letters indicate significant differences (p< 0.05) between groups; asterisks indicate significance (p<0.05) relative to untreated controls.

Article Snippet: The phosphorylation of protein kinases in CD14+ monocytes treated with late-stage OA SF for 1 hour was assessed using the Human Phospho-Kinase Array Kit (R&D Systems; ARY003C), according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Cell Culture, Gene Expression

Journal: bioRxiv

Article Title: Proteinase 3 is involved in presepsin production through neutrophil extracellular trap phagocytosis by macrophages

doi: 10.1101/2025.05.02.651854

Figure Lengend Snippet: The percentage of the neutrophil extracellular trap (NET) area in the SS and FS plots of flow cytometry was compared among untreated neutrophils and samples following NET induction with E. coli DH5α or PMA. (a) Expression intensities of Cit-H3, flavocytochrome b558, SYTOX™ Green, CD14, TLR2, TLR4, and LL-37 were compared between untreated neutrophil and NET areas after E. coli DH5α stimulation. (b) NET ratios were compared among untreated neutrophils and samples treated with E. coli DH5α or PMA using enzyme-linked immunosorbent assay (ELISA). Data are presented as mean ± standard deviation (SD) (n = 3). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. ** p < 0.01. (c) Western blotting was performed to examine CD14 and Cit-H3 protein levels in untreated neutrophils and those stimulated with E. coli DH5α or PMA. Band intensities were quantified using ImageJ and normalized to β-actin. Data are presented as mean ± standard deviation (SD) (n = 3). Data are presented as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey–Kramer’s HSD test. ** p < 0.01

Article Snippet: Details of the reagents used in this study and their sources are as follows: Polymorphprep ™ (Cat. No.1114683; Axis-Shield, Dundee, Scotland), RPMI-1640 Medium (Cat. No. R8758; Sigma-Aldrich, MO, USA), phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, MO, USA), Escherichia coli DH5α competent cells ( E. coli DH5α; Cat. No. 9057; Takara Bio, Shiga, Japan), EasySep ™ Human Monocyte Isolation Kit (Cat. No. 19359; Stemcell Technologies, Vancouver, BC, Canada), CellXVivo TM Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep Permeabilizaton Reagent (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), cytochalasin D (Cat. No. 11330; Cayman Chemical, Ann Arbor, MI, USA), wortmannin (Cat. No. AG-CN2-0023-M001; Adipogen Life Sciences, San Diego, CA, USA), phenylmethylsulfonyl fluoride (PMSF; Cat. No. 195381; MP Biomedicals, Irvine, CA, USA), elafin (Cat. No. 4243-v; Peptide Institute, Osaka, Japan), May–Grünwald’s stain solution (Cat. No. 15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No. 15002; Mutokagaku, Tokyo, Japan), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No. 15612; Mutokagaku, Tokyo, Japan), 4% paraformaldehyde phosphate buffer solution (Cat. No. 09154-85; Nacalai Tesque, Kyoto, Japan), Triton ™ X-100 (Cat. No. 194854; MP Biomedicais, Irvine, CA, USA), sample buffer solution with 2-mercaptoethanol (Cat. No. 30566-22; Nacalai Tesque, Kyoto, Japan), skim milk powder (Cat. No. 190-12865; Fujifilm Wako Pure Chemical, Tokyo, Japan), Chemi-Luna One Super (Cat. No. 02230; Nacalai Tesque, Kyoto, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Tokyo, Japan), SYTOX ™ Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechest ® 33342 (Cat. No. 346-07951; Fujifilm, Tokyo, Japan), Recombinant human CD14 protein (rCD14; Cat. No. 383-CD; R&D systems, Minneapolis, MN, USA), and recombinant human PR3 protein (Cat. No. 13699-H08H1; Sino Biological, Kanagawa, Japan).

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Western Blot

Journal: bioRxiv

Article Title: Proteinase 3 is involved in presepsin production through neutrophil extracellular trap phagocytosis by macrophages

doi: 10.1101/2025.05.02.651854

Figure Lengend Snippet: (a) Presepsin (P-SEP) levels in the supernatant were measured after addition of PR3 to rCD14. Conditions with PR3 inhibition using the inhibitors phenylmethylsulfonyl fluoride or elafin were also compared. (b) Western blot analysis of CD14 and P-SEP proteins, with band intensities quantified using ImageJ. Data are presented as mean ± SD (n = 3). Statistical analysis was performed using one-way analysis of variance followed by Tukey–Kramer’s HSD test. ** p < 0.01.

Article Snippet: Details of the reagents used in this study and their sources are as follows: Polymorphprep ™ (Cat. No.1114683; Axis-Shield, Dundee, Scotland), RPMI-1640 Medium (Cat. No. R8758; Sigma-Aldrich, MO, USA), phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, MO, USA), Escherichia coli DH5α competent cells ( E. coli DH5α; Cat. No. 9057; Takara Bio, Shiga, Japan), EasySep ™ Human Monocyte Isolation Kit (Cat. No. 19359; Stemcell Technologies, Vancouver, BC, Canada), CellXVivo TM Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep Permeabilizaton Reagent (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), cytochalasin D (Cat. No. 11330; Cayman Chemical, Ann Arbor, MI, USA), wortmannin (Cat. No. AG-CN2-0023-M001; Adipogen Life Sciences, San Diego, CA, USA), phenylmethylsulfonyl fluoride (PMSF; Cat. No. 195381; MP Biomedicals, Irvine, CA, USA), elafin (Cat. No. 4243-v; Peptide Institute, Osaka, Japan), May–Grünwald’s stain solution (Cat. No. 15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No. 15002; Mutokagaku, Tokyo, Japan), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No. 15612; Mutokagaku, Tokyo, Japan), 4% paraformaldehyde phosphate buffer solution (Cat. No. 09154-85; Nacalai Tesque, Kyoto, Japan), Triton ™ X-100 (Cat. No. 194854; MP Biomedicais, Irvine, CA, USA), sample buffer solution with 2-mercaptoethanol (Cat. No. 30566-22; Nacalai Tesque, Kyoto, Japan), skim milk powder (Cat. No. 190-12865; Fujifilm Wako Pure Chemical, Tokyo, Japan), Chemi-Luna One Super (Cat. No. 02230; Nacalai Tesque, Kyoto, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Tokyo, Japan), SYTOX ™ Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechest ® 33342 (Cat. No. 346-07951; Fujifilm, Tokyo, Japan), Recombinant human CD14 protein (rCD14; Cat. No. 383-CD; R&D systems, Minneapolis, MN, USA), and recombinant human PR3 protein (Cat. No. 13699-H08H1; Sino Biological, Kanagawa, Japan).

Techniques: Inhibition, Western Blot

Journal: bioRxiv

Article Title: Proteinase 3 is involved in presepsin production through neutrophil extracellular trap phagocytosis by macrophages

doi: 10.1101/2025.05.02.651854

Figure Lengend Snippet: Upon bacterial infection of blood vessels, neutrophils release neutrophil extracellular traps (NETs) as a host defense mechanism. M1 MΦs phagocytose CD14-rich NETs, and the engulfed CD14 is degraded by the proteolytic enzyme PR3 within the macrophages, resulting in the generation of presepsin (P-SEP). Subsequently, P-SEP is released extracellularly from M1 MΦs, leading to elevated blood levels of P-SEP.

Article Snippet: Details of the reagents used in this study and their sources are as follows: Polymorphprep ™ (Cat. No.1114683; Axis-Shield, Dundee, Scotland), RPMI-1640 Medium (Cat. No. R8758; Sigma-Aldrich, MO, USA), phorbol 12-myristate 13-acetate (PMA; Cat. No. P8139; Sigma-Aldrich, MO, USA), Escherichia coli DH5α competent cells ( E. coli DH5α; Cat. No. 9057; Takara Bio, Shiga, Japan), EasySep ™ Human Monocyte Isolation Kit (Cat. No. 19359; Stemcell Technologies, Vancouver, BC, Canada), CellXVivo TM Human M1 Macrophage Differentiation Kit (Cat. No. CDK012; R&D systems, Minneapolis, MN, USA), IntraPrep Permeabilizaton Reagent (Cat. No. A07803; Beckman Coulter, Brea, CA, USA), cytochalasin D (Cat. No. 11330; Cayman Chemical, Ann Arbor, MI, USA), wortmannin (Cat. No. AG-CN2-0023-M001; Adipogen Life Sciences, San Diego, CA, USA), phenylmethylsulfonyl fluoride (PMSF; Cat. No. 195381; MP Biomedicals, Irvine, CA, USA), elafin (Cat. No. 4243-v; Peptide Institute, Osaka, Japan), May–Grünwald’s stain solution (Cat. No. 15053; Mutokagaku, Tokyo, Japan), Giemsa stain solution (Cat. No. 15002; Mutokagaku, Tokyo, Japan), 1/15 M phosphate buffer solution (pH 6.4) (Cat. No. 15612; Mutokagaku, Tokyo, Japan), 4% paraformaldehyde phosphate buffer solution (Cat. No. 09154-85; Nacalai Tesque, Kyoto, Japan), Triton ™ X-100 (Cat. No. 194854; MP Biomedicais, Irvine, CA, USA), sample buffer solution with 2-mercaptoethanol (Cat. No. 30566-22; Nacalai Tesque, Kyoto, Japan), skim milk powder (Cat. No. 190-12865; Fujifilm Wako Pure Chemical, Tokyo, Japan), Chemi-Luna One Super (Cat. No. 02230; Nacalai Tesque, Kyoto, Japan), Cell Death Detection ELISAPLUS (Cat. No. 11774425001; Roche Diagnostics, Tokyo, Japan), SYTOX ™ Green (Cat. No. S7020; Invitrogen, Waltham, MA, USA), Hoechest ® 33342 (Cat. No. 346-07951; Fujifilm, Tokyo, Japan), Recombinant human CD14 protein (rCD14; Cat. No. 383-CD; R&D systems, Minneapolis, MN, USA), and recombinant human PR3 protein (Cat. No. 13699-H08H1; Sino Biological, Kanagawa, Japan).

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: Metabolomic and immune alterations in long COVID patients with chronic fatigue syndrome

doi: 10.3389/fimmu.2024.1341843

Figure Lengend Snippet: Elevated levels of proinflammatory biomarkers in LC patients. (A) Cumulative data comparing the plasma IL-1α, (B) IL-6, (C) TNF-α, (D) IP-10, (E) CRP, (F) SAA, (G) IL-13, and (H) IL-1β, (I) Flt-1, and (J) soluble CD14 measured by ELISA in the plasma of R, LC, and HC groups. (K) Comparing the Anti-CaSR antibody levels in plasma samples of HC, R, and LC group. (L) Soluble CaSR levels in plasma samples of HC, R, LC and acute COVID-19 patients. (M) Cumulative data of the plasma ATP in HC, acute, R, and LC groups. Kruskal–Wallis analysis with Dunn’s multiple comparisons test. ns, not significant. Each dot represents a study subject. * p < 0.5, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: The plasma was subjected to ELISA kit sCD14 (R&D, 383CD-050) ( ) and Anti-CaSR (EAGLE Biosciences).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Journal of microbiology and biotechnology

Article Title: Anti-Inflammatory Efficacy of Human-Derived Streptococcus salivarius on Periodontopathogen-Induced Inflammation.

doi: 10.4014/jmb.2302.02002

Figure Lengend Snippet: Fig. 6. Binding inhibition of LPS or LTA of periodontopathogens to CD14 and LBP by S. salivarius LTA. EIA plates were coated with rhCD14 (A-C) and rhLBP (D-F). The plates were incubated with biotinylated P. gingivalis LPS (100 ng/ml), T. forsythia LPS (100 ng/ml), and F. alocis LTA (100 ng/ml) in the presence or the absence of S. salivarius G7 LTA and type strain LTA. The bound LPS and LTA were detected using HRP-labelled streptavidin and TMB solution. The experiments were performed three times in duplicate, and data are represented as means and standard deviations. Asterisk (*) indicates statistically significant differences compared with non-treated group (p < 0.05).

Article Snippet: Recombinant human LBP (rhLBP, R&D Systems) and human CD14 (rhCD14, R&D Systems) were dissolved in DPBS at a concentration of 2 μg/ml and were added into suitable Ab-coated well.

Techniques: Binding Assay, Inhibition, Incubation