16-0149-82

antibody from Invitrogen Antibodies

Targeting: CD14

Provider product page for 16-0149-82

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Antibody Data
Antigen structure
References [44]
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Flow cytometry [1]
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Product number: 16-0149-82 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD14 Monoclonal Antibody (61D3), Functional Grade, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The 61D3 monoclonal antibody reacts with human CD14, a 53-55 kDa GPI-linked glycoprotein. CD14 is expressed on monocytes, interfollicular macrophages and some dendritic cells. Complexes of LPS and LBP (LPS-Binding Protein) bind with high affinity to monocytes through the surface CD14. Applications Reported: The 61D3 antibody has been reported for use in flow cytometric analysis. 61D3 has also been reported for in vitro functional studies. (Fluorochrome-conjugated 61D3 is recommended for use in flow cytometry). Applications Tested: The 61D3 antibody has been tested by flow cytometric analysis of normal human peripheral blood cells. This can be used at less than or equal to 1 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Storage and handling: Use in a sterile environment. Filtration: 0.2 µm post-manufacturing filtered. Purity: Greater than 90%, as determined by SDS-PAGE. Endotoxin Level: Less than 0.001 ng/µg antibody, as determined by LAL assay. Aggregation: Less than 10%, as determined by HPLC.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: 61D3
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: 4° C

CD14 protein structure - 16-0149-82 shown in red.

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Experimental details: Figure 6 Representative high-magnification photomicrographs showing double immunofluorescence for NAAA, CD19, CD3 and CD14 in order to characterize the immune cells in the mucosa infiltrate of UC patients. NAAA immunofluorescence was observed in CD19+ B lymphocytes (A-C), CD3+ T lymphocytes (D-F) and CD14+ macrophages (G-I).

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Experimental details: Fig. 1 Isolation and characterization of huglia/HIV (HC69) cells. a Schematic representation of a typical procedure to develop a microglia/HIV clonal cell population such as huglia/HIV (HC69) cells. Uninfected clonal populations are indicated in grey boxes , and latently-infected clonal populations are indicated in blue boxes . b Immunofluorescence analysis of the human microglial cells huglia/HIV (HC01). Cells were cultured, fixed, and immunostained with either anti-CD11b ( green ), anti-CD14 ( red ) or anti-P2RY12 ( red ) conjugated antibodies. Nuclei were stained with DAPI ( blue ). Merged images of nuclei, CD11b and CD14, or nuclei, CD11b and P2RY12 are indicated

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Experimental details: 1 P. gingivalis minor fimbriae-mediated invasion drives optimal DC differentiation. CD14 + monocytes were treated with P. gingivalis strains DPG-3 (mfa-1 + ) and MFB (mfa1 - fimA - ) or the DC-SIGN ligand HIV gp120. Cells were analyzed by flow cytometry to determine differentiation by loss of CD14 and gain of DC-SIGN. (A) The DPG-3-treated cells show an up-regulation of DC-SIGN significantly higher than conventional MoDCs at 1 h and 24 h, where the other treatments do not. Monocytes pretreated with gp120 show a significant decrease in DC-SIGN at 24 h. (B) DPG-3-treated monocytes significantly down-regulate the monocyte marker CD14 compared with conventional MoDCs at 1 h and 24 h, whereas other treatments cause a down-regulation of CD14 at 1 h but no significant decreases at 24 h. T P

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Experimental details: 2 P. gingivalis mfa-1 fimbriae, not fimA, drive rapid DC differentiation. (A and B) CD14 + monocytes were treated with DPG-3 (mfa-1 + fimA - ) and MFI (mfa-1 - fimA + ) or the DC-SIGN ligand gp120 prior to DPG-3 infection. Cells were analyzed by flow cytometry to determine differentiation by loss of CD14 and gain of DC-SIGN. (A) Only DPG-3-treated monocytes display a significant increase of DC-SIGN. MFI does not drive a rapid increase of DC-SIGN expression, and blocking DC-SIGN prevents DPG-3 from causing a significant up-regulation of DC-SIGN. (B) MFI causes less CD14 down-regulation than DPG-3 at 1 h, and MFI and gp120 treatments have significantly higher CD14 expression at 6 h. (C and D) Monocytes are treated with DC-SIGN-targeting siRNA or scrambled siRNA fragments simultaneously with bacterial infections. (C) Disruption of DPG-3 uptake by siRNA at 6 h prevents up-regulation of DC-SIGN driven by DPG-3. At 24 h, only DPG-3-infected or DPG-3 and off-target (OT) controls show a significant increase in DC-SIGN expression. (D) Only DPG-3-treated monocytes have significantly decreased CD14 at 6 h, and all DPG-3-infected samples show a significant decrease of CD14 by 24 h. T P

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Experimental details: 3 PDDCs have intact antigen-capture function. CD14 + monocytes were treated with CFSE-labeled mutant strains of P. gingivalis for 24 h to generate PDDC. Inactivated, whole E. coli were fluorescently labeled and used to measure phagocytosis of CFSE-positive PDDC by flow cytometry. All of the PDDCs were highly phagocytic compared with conventional MoDCs. MFI-generated PDDCs were less phagocytic than the other treatments but still significantly higher than conventional MoDCs. T P

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Experimental details: Figure 2 Cell surface expression of SAIL in CLL, AML and MM patient samples and normal BMMC and PBMC controls. ( a ) Three CLL specimens analyzed by flow cytometry. CLL cells were identified as CD19/CD5 double-positive cells. The histograms present SAIL (filled) and isotype control (open) staining in the live-cell and the CLL population. ( b ) Flow cytometry analysis of three AML specimens. SAIL expression is assessed in live-cells, CD33-positive and CD34-positive cells. ( c ) Flow cytometry analysis of three MM specimens. CD38 high cells with CD56 expression were gated for MM cells. SAIL expression is assessed in the live-cell and the MM population. ( d and e ) Flow cytometry analysis of SAIL expression in BMMC ( d ) and PBMC ( e ) via co-staining with CD19, CD3, CD14, CD56, CD33, CD34 and a cocktail of lineage (LN) markers. Numbers in histograms are median-fluorescence-intensity fold-change values relative to the isotype control. Three and two representative examples are shown for the tumor and normal samples, respectively.

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Experimental details: Figure 2 PD-1 and PD-L1 were upregulated on T cells and monocytes in septic shock patients . Blood samples were obtained from 19 septic shock patients and 22 healthy controls and were stained for programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) gated on CD4 + T cells, CD8 + T cells, and CD14 + monocytes. (a) to (c) Percentage of PD-1 expression on (a) CD4 + T cells and (b) CD8 + T cells, and (c) percentage of PD-L1 expression on CD14 + monocytes. Each dot represents one individual. Data are mean +- standard error of the mean (SEM) of three independent experiments. ** P < 0.01 compared with healthy controls. (d) to (f) Mean fluorescence intensity (relative fluorescence units) of PD-1 expression on (d) CD4 + T cells, (e) PD-1 expression on CD8 + T cells, and (f) PD-L1 expression on CD14 + monocytes Each dot represents one individual. Data are mean +- SEM of three independent experiments. * P < 0.05 compared with healthy controls. (g) Representative PD-1 expression levels on CD4 + T cells and CD8 + T cells, and PD-L1 expression on CD14 + monocytes. Values in the upper-right quadrant indicate the percentage of cells that express PD-1 or PD-L1.

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Experimental details: Fig 7 Measurement of ROS in CD14 + cells, CD4 + T-cells, and CD8 + T-cells by cellROX stain mean intensity in T2DM patients compared to healthy. CD14 + cells were stained with cellROX green reagent, a marker of ROS, and a CD14 cell marker, CD14-PE. CD14 + -ROX + cells' mean intensity was analyzed by FLOW cytometry. There was an observable increase in ROX mean intensity in CD14 + cells isolated from individuals with T2DM compared to healthy volunteers (Fig. 7A). CD4 + cells were also stained with cellROX green reagent and a CD4 cell marker, CD4-Cy5. CD4 + -ROX + cells' mean intensity was analyzed by FLOW cytometry. There was an observable increase in ROX mean intensity in CD4 + T-cells isolated from individuals with T2DM compared to healthy volunteers (Fig. 7B). CD8 + cells were stained with cellROX green reagent and a CD8 cell marker, CD8-Cy5. CD8 + -ROX + cells' mean intensity was analyzed by FLOW cytometry. There was an observable increase in ROX mean intensity in CD8 + T-cells isolated from individuals with T2DM compared to healthy volunteers (Fig. 7C). Data represents mean +-SE from 5 healthy individuals and 5 individuals with T2DM.

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Experimental details: FIG. 2. Baseline comparison of the interleukin-6 (IL-6) and reactive oxygen species (ROS) markers between healthy volunteers and HIV-positive individuals. We observed a significant increase in the levels of the proinflammatory cytokine, IL-6 in plasma samples collected from individuals with HIV infection compared to healthy individuals (A) . Data represent mean+-SE from comparing baseline levels of 10 healthy volunteers and 15 HIV-positive individuals, **** P

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Experimental details: FIG. 6. Difference in plasma IL-6 levels and ROS markers pre- and post-GSH supplementation. Sandwich ELISA was performed to compare the cytokine levels between pre-supplementation (V1) and post-supplementation (V3). Assay of cytokines showed a significant decrease in the levels of IL-6 in plasma samples collected from the lGSH-treatment group. There was no significant difference between the levels of IL-6 from the placebo group when comparing visit 1 and visit 3 (A) . Data represent mean+-SE, * P

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Experimental details: Figure 1 Effect of OxC-beta on CD14, TLR-4, and TLR-2 levels in vitro . Human THP-1 monocytes (A), fibroblasts (B), and endothelial cells (C), were treated with the indicated concentrations of OxC-beta or vehicle control (DMSO) for 24 hours. Immune receptor content was measured 24 hours post-treatment by FACS analysis. OxC-beta-induced increase in receptor level was assessed relative to untreated control cells using a one-way analysis of variance with Tukey's post test for multiple comparisons. DMSO had no effect on receptor level (result not shown). Phorbol myristate acetate (PMA) was used as a positive control in experiments with THP-1 cells (hatched bars).

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Experimental details: Figure 2 CD14 and TLR-4 staining in gut epithelial cells. Balb/c mice were not supplemented (control) or supplemented daily by oral gavage with OxC-beta (10 mg/kg). After 4 weeks, intestinal tissues were harvested and CD14 and TLR-4 expression was determined by immunocytochemistry. Increased CD14 (A) and TLR-4 (C) expression is readily apparent in epithelial cells in the OxC-beta-supplemented animals compared to the controls receiving vehicle alone (B and D, respectively). Arrows indicate the location of enterocytes within the cross section of microvilli. Magnification 40x.

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Experimental details: Figure 5 Determination of activities relative to OxC-beta of (A) OxC-beta polymer and monomer fractions, and (B) oxidized lycopene (OxC-lyc), using a CD14 receptor expression assay. THP-1 cells were treated for 24 hours with the indicated concentrations of compounds. CD14 expression was quantified using FACS analysis. The effect of each compound is shown relative to untreated cells. Points represent the mean and standard error from three separate experiments. (A) Correlation analysis indicates a significant dose effect for each compound on CD14 expression with p-values of 0.0036 for OxC-beta, 0.0034 for the polymer, and 0.0113 for the monomer. Comparison of the relative activity of each compound indicates that the monomer is significantly less active than the polymer (p

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Experimental details: Figure 1 PMA-induced TM expression mediates morphological changes and differentiation marker expression in THP-1 cells. ( A ) THP-1 cells were treated with 150 nM PMA for 24-72 hours. The total cell lysates were harvested, and the expression of TM was analyzed using western blot analysis. beta-actin was used as a loading control. Five independent experiments have been performed (n = 5) and representative images have been showed. The amount of proteins expression was quantified using densitometry and presented as bar graph. The data are presented as the mean +- SD (n = 5), and * p < 0.05 was considered significant. ( B ) THP-1 cells were transfected with TM siRNA or HA-TM FL plasmid for 24 h followed by PMA stimulation for 72 hours. The morphology of the cells was observed using light microscopy. The adherent differentiated macrophage-like cells are indicated by a white arrowhead. Five independent experiments have been performed (n = 5). The quantification is shown in the right graph. ( C ) The expression of the macrophage cell surface markers CD14 (red) and CD68 (green) was analyzed using immunofluorescence and microscopy. Hoechst staining was used to label the nuclei. The scale bar indicates 100 mum. Five independent experiments have been performed (n = 5), and showed representative images. ( D ) The expression of CD14 and CD68 was analyzed using flow cytometry. Data are expressed as a % of the control, are presented as the mean +- SD and represent the results of three indep

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Experimental details: Figure 4 TM regulates THP-1 cell differentiation via the PKCdelta-ERK1/2 signaling pathway. ( A ) THP-1 cells were transfected with 2 or 4 mug of PKCalpha, PKCbeta, PKCdelta, PKCepsilon, or PKCtheta shRNA for 24 h. Total cell lysates were purified, and knockdown efficiency was assayed using western blot analysis. ( B ) The THP-1 cells were knocked down by PKCalpha, PKCbeta, PKCdelta, PKCepsilon, and PKCtheta shRNAs for 24 h followed by PMA stimulation for 72 hours. The number of CD14 + cells was scored using flow cytometry. Data are expressed as a % of the control, are presented as the mean +- SD and represent the results of five independent experiments (n = 5, * p < 0.05 was considered significant). ( C ) Different sets of THP-1 cells were transfected with 4 mug of each shRNA for 24 h followed by PMA stimulation for 72 hours. The levels of p21 Cip1/WAF1 and PCNA were analyzed using western blot analysis. ( D ) The THP-1 cells were knocked down by PKCdelta shRNA for 24 h followed by PMA stimulation for 72 hours. The level of PKCdelta and total and phosphorylated ERK1/2 was analyzed using western blot analysis. In western blot analysis, beta-actin and total-ERK1/2 were used as loading controls. The density of each band was quantified using densitometry and related protein expression was presented as bar graph. The data are presented as the mean +- SD, and * p < 0.05 was considered significant (n = 5). ( E ) Lysates of THP-1 cells with PMA stimulation were extracted. Left, immu

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Experimental details: Fig. 1 Flow cytometry analysis of phenotype characterization of hUCMSCs. Phenotype of CD73, CD90, CD105, CD14, CD34, CD45, CD79a and HLA-DR of hUCMSCs was detected by flow cytometry. Intensity >= 95% represented strong expression while

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Experimental details: Figure 1. Immunophenotypic profile of cultured cartilage endplate-derived stem cells. The blue lines represent the fluorescence intensity of cells stained with the indicated antibodies and the red lines represent the negative controls cells, which were stained with a non-immunoreactive isotype control antibody. FITC, fluorescein isothiocyanate; PerCP, peridinin chlorophyll; PE, phycoerythrin; HLA-DR, human leukocyte antigen-antigen D related.

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Experimental details: Fig. 1 Continuous generation of human iPSC-Mac in stirred tank bioreactors. a Scheme of hematopoietic differentiation of human iPSC in stirred tank bioreactors (DASbox system). b Representative pictures of DASbox bioreactor filled with floating MCFCs (left). Images of the 8-blade impeller (right). c Individual cell counts of viable macrophages produced in bioreactors ( n = 2 of independent bioreactor runs, mean +- SD.). d Representative macrophage harvest counts (upper graph) and corresponding data from continuous process monitoring (biomass, temperature, pH, and dissolved oxygen (DO) level) for the entire cultivation phase of one 42-day bioreactor run. e Analysis of human cytokines in the medium from the bioreactor (technical duplicates, mean +- SD). f Representative light microscopy of macrophage generation analyzed at harvests 1-5. First row shows brightfield images of non-filtered medium samples from bioreactors (scale bar 500 um). Second row shows brightfield images of freshly harvested macrophages separated from MCFCs by sedimentation (scale bar 50 um). Third and fourth row show cytospin (scale bar 50 um) and flow cytometric analysis of iPSC-Mac (gray line: respective isotype control, red line: CD45, blue line CD14)

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Experimental details: Figure 2. CD3+CD14+ cells are tightly bound T cell:monocyte complexes that represent in vivo association. ( A ) Gating strategy and ( B ) random gallery of events for monocytes (CD14+CD3), T cells (CD3+CD14-), CD3+CD14hi cells and CD3+CD14mid cells determined by imaging flow cytometry (ImageStreamX, MkII Amnis Amnis). CD14+ cell debris were identified within CD3+CD14mid cells ( C ) by imaging flow cytometry and ( D ) confocal microscopy after bulk population cell sorting. ( E ) Plots and (F) Ratio of Aspect ratio vs Area of the brightfield parameter for monocytes (CD14+CD3-), T cells (CD3+CD14-), CD3+CD14hi cells and CD3+ CD14 mid cells, determined by imaging flow cytometry. ( G ) Non-parametric Spearman correlation of the frequency of T cell:monocyte complexes in paired fresh PBMC vs cryopreserved PBMC derived from 45 blood draws of healthy subjects. T cell:monocyte complexes were defined as the CD3+CD14+ cell population gated from live singlets as represented in Figure 1-figure supplement 2 . ( H ) Single z-plan (0 mum) images ( left ) and z-plane stacks ( right ) of the region marked (dashed rectangle) from one sorted CD3+ CD14+ T cell:monocyte complex displaying accumulation of LFA1 and ICAM1 at the interface. Images show expression of CD14 (blue), CD3 (green), ICAM1(Cyan), and LFA1 (Magenta). Relative z-positions are indicated on the right, and scale bars represent 2 mum. Imaging flow cytometry data was derived from 10 subjects across three independent experiments and mi

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Experimental details: Figure 1 M1 macrophage and STAT1 were excessive in RSA patients. ( A-B ) The dot plot represents labeling of CD14 + CD86 + and CD14 + TNFalpha + (M1) cells by flow cytometry in decidua of NP subjects (n= 30) and RSA patients (n= 30). ( C ) qRT-PCR analysis of STAT1 expression in the decidua of NP subjects (n= 30) and RSA patients (n= 30). ( D ) STAT1 and p-STAT1 protein levels were measured in decidua of NP subjects (n= 10) and RSA patients (n= 10) by western blot. ( E ) Representative IHC staining images of STAT1 in the decidua of NP and RSA patients (Scale bar, 50 um, 200x). ( F ) Correlation between p-STAT1 and the proportion of CD14 + CD86 + in decidua of NP subjects (n= 10) and RSA patients (n= 10). ( G ) Correlation between p-STAT1 and the proportion of CD14 + TNF-alpha + in decidua of NP subjects (n= 10) and RSA patients (n= 10). Values were listed as the mean+- SEM. **** P < 0.0001.

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Experimental details: Fig. 1 Identification of EPCs. a Adherent cells grew in a blood island manner. Fluorescent staining of EPCs. b Adherent cells took up UEA-1-lectin. c Adherent cells took up Dil-Ac-LDL. d Adherent cells took up UEA-1-lectin and Dil-Ac-LDL. E. Surface molecular markers of EPCs. Adherent cells expressed CD34, CD133, CD14 and VEGFR-2. All experiments involving cell culture studies were repeated three times with three replicates per experiment

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Experimental details: 10.1080/19420862.2020.1725365-F0003 Figure 3. Antibody-dependent cellular cytotoxicity (ADCC) (a) and antibody-dependent cellular phagocytosis (ADCP) (b) of the antibodies against CTLA-4. (a) Human CTLA4-expressing 293F cells were added to 96-well plates at 1 x 10 4 cells/well, and then the antibodies pre-incubated with 5 x 10 5 PBMCs were added. The plates were kept at 37degC in a 5% CO 2 incubator for 4 h. Lysis of the target cells was determined by the introduction of DELFIA(r) EuTDA Cytotoxicity Reagents. (b) Human macrophage cells were mixed at 1:1 ratio with CFSE-dyed engineered human CTLA-4 expressing 293F cells in 96-well plates, then antibodies were added and incubated with cells at 37degC in a 5% CO 2 incubator for 3 h. After wash, APC-labeled anti-human CD14 antibody was added for flow cytometry detection.

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Experimental details: Figure 2 Comparison of monocytes and NK cell percentage amongst study groups. A. The stained PBMCs were gated on the monocyte population and CD3 + CD19 + cells were excluded. Cell populations are displayed for CD16 and CD14 expression (upper FACS panel). One exemplary dot plot is shown per study group. The bar diagrams (lower panel) show the non-classical (CD16 + CD14 - ), intermediate (CD16 + CD14 + ) and classical (CD16 - CD14 + ) monocytes. *P-value

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Experimental details: Fig. 4 GAPDH exposed on apoptotic cells and phagocyte CD14 mediate efferocytosis. A Co-localization of apoptotic cell surface GAPDH with CD14 on phagocyte. When cells were incubated together the two signals co-localize at points of intercellular contact. Scale bar, 5 mum. B Co-immunoprecipitation (Co-IP) of GAPDH from mixed membrane fractions of apoptotic cells (J774) and phagocytes (THP-1). Right side panels are Western blots of membrane fractions as positive control for both antibodies. C Western blot to confirm the CD14 K/D in THP-1 cells. Cell lysates were prepared from THP-1 empty vector and CD14 knockdown cells and samples were run on 10% SDS-PAGE. Separated proteins were transblotted onto PVDF membrane and probed with mouse anti-CD14 antibody (Abcam) followed by secondary antibody anti-mouse peroxidase (Sigma). D - E Phagocytosis of apoptotic cells is dependent upon both GAPDH on apoptotic cell surface and CD14 on phagocytes. D Representative confocal microscopy images of live and apoptotic J774 cells that were phagocytosed by THP-1 phagocytes. Live cells, apoptotic empty vector, and GAPDH K/D J774 cells are labeled with Vybrant DiD dye (Red). Phagocyte THP-1 cells, empty vector, and CD14 K/D cells are labeled with CFSE (Green). Scale bar, 20 mum. E Bar graph represents phagocytosis as percentage of control (extent of phagocytosis by THP-1 empty vector cells incubated with J774 empty vector cells as 100%), *** P < 0.0001, n = 150 cells, also see Fig. S2C-D . F Disrupti

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Experimental details: Fig. 7 IRF5 binds to GHITM and regulates mitochondrial activity in human monocytes and adipose tissue macrophages. a CD14 + Monocytes from patients with type-2 diabetes (T2D; n = 5) were sorted based on expression of IRF5 for RNA-seq. Differential analyses were paired by patient and carried out on IRF5 + versus IRF5 - monocytes ( n = 5, Wald test p -value < 0.05). b Gene ontology (GO) term enrichment from upregulated and downregulated genes in IRF5 + versus IRF5 - monocytes. c Expression of Ghitm in IRF5 - and IRF5 + monocytes ( n = 5, * p = 0.039, two-tailed paired t -test). d Irf5 and Ghitm counts in white adipose tissue (WAT) macrophages and monocytes, from public dataset of scRNA-seq of the stromal vascular fraction (SVF) of patients that are lean or with obesity (two-tailed unpaired t -test, **** p < 0.0001). Heatmap of single-cell expression of Irf5 and Ghitm from monocytes (Mon) and macrophages (Mac), each line represents a single cell. e Proportion of Ghitm + (blue) and Ghitm- (red) cells in 10-cell bins by increasing Irf5 expression. f Correlation of Ghitm and Irf5 mean expression per bin (Pearson's correlation Pearson R 2 = 0.28, two-tailed p < 0.0001). g Ghitm expression in CD14 + human visceral adipose tissue macrophages (vATMs). Samples were stratified based on expression of Irf5 into IRF5 Lo versus IRF5 Hi expressors (IRF5 Lo n = 7 and IRF5 Hi n = 6, two-tailed unpaired t -test, p = 0.13). h Correlation of IRF5 MFI, JC1-Green (mitochondrial mass, Mt Mass), JC1-R

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Experimental details: Figure 2 Induced differentiation, flow cytometric and immunosuppressive ability analysis of human umbilical cord mesenchymal stem cells expanded in serum-free medium. After differentiation induction, (a) osteogenesis was confirmed by Alizarin Red (x40), (b) adipogenesis was stained by Oil Red O (x200) and (c) chondrogenesis was analyzed by Toluidine Blue (x100). (d) Serum-free medium (SFM)-expanded human umbilical cord mesenchymal stem cells (hUC-MSCs) at the 10th passage were labeled with antibodies against human antigens CD14-PE, CD19-PE, CD34-FITC, CD45-PE, CD73-PE, CD90-PE, CD105-PE, HLA-ABC-FITC, HLA-DR-PE and Nestin-PE. (e) Expression of hTERT in hUC-MSCs. Graph shows the level of hTERT transcripts of hUC-MSCs cultured in serum-containing medium (SCM) and SFM ( n = 5). Values presented as ratio of positive control (HeLa cells). Immunosuppressive ability of hUC-MSCs was evaluated by co-culturing with human peripheral blood mononuclear cells (hPBMCs). (f) Proliferation of hPBMCs was quantified based the measurement of BrdU incorporation during DNA synthesis. (g) Level of interferon gamma (IFN-gamma) in the supernatant was determined by ELISA.

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Experimental details: Fig. 2 a Cumulative population-doubling (cPD) levels versus passage number for the four different sources of MSC. Black represents CV-MSC ( n = 7), dark gray UC-MSC ( n = 4), medium gray AT-MSC ( n = 5), and light gray BM-MSC ( n = 6). b IHC-based senescence-associated beta-galactosidase (SA-beta-gal) staining of CV-MSC in early (i, passage 4) and late (ii, passage 9) passages, AT-MSC in passage 6 (iii), BM-MSC in passage 6 (iv), and UC-MSC in passage 2 (v) and passage 4 (vi). Scale = 200 mum. c IHC of CV-MSC (i, ii) and BM-MSC (iii, iv) stained for osteopontin (i, iii) and fibronectin (ii, iv). Scale = 1 mm. d Collagen area (%) after collagen contraction assay for CV-MSC ( n = 4), BM-MSC ( n = 3), UC-MSC ( n = 4), and AT-MSC ( n = 3). Cells in passage 3 were used. Results expressed as mean +- SD, percentage of the total collagen area of the collagen gels without cells. e Surface marker expression of CV-MSC in early passages ( n = 5). Results expressed as mean +- SD (%). f Representative immunofluorescence of early passaged CV-MSC (i, iii) and BM-MSC (iii, iv) stained for SM22alpha (i, iii) and alpha-SMA (ii, iv). Scale = 50 mum. AT adipose tissue, BM bone marrow, CV chorionic villi, MSC mesenchymal stromal cells, UC umbilical cord

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Experimental details: Fig. 5 KDM6B promotes the differentiation of THP-1 cells (A) THP-1 cells were treated with 100 ng/ml PMA, 2 muM GSK-J4, or DMSO for 48 h. CD14 and CD11b expression levels were confirmed using qRT-PCR and normalized to GAPDH . Results are shown as mean +- SEM, n = 3; *p < 0.05, **p < 0.01. (B) We treated negative control (shNC)- and shKDM6B-transfected THP-1 cells with 100 ng/ml PMA for 48 h. The cells were stained with PE-CD11b and APC-CD14 antibodies. The percentage of cells in each quadrant is indicated in the figure. (C) We treated THP-1 cells with 2 muM GSK-J4 or 0.3 muM alisertib for 48 h. The cells were stained with PE-CD11b and APC-CD14 antibodies. The percentage of cells in each quadrant is indicated in the figure. (D) A model of AURKA regulating KDM6B expression in PMA-mediated THP-1 differentiation.

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Experimental details: Figure 4. Butyrate upregulates the levels of TLR4 and CD14 on colon cancer cells. The expression levels of TLR4 and CD14 on the membrane of SW480 cells and CT26 cells treated by butyrate and/or LPS were (A) analyzed using a flow cytometer, and (B) the MFI values of TLR4 and CD14 were quantitative analyzed. These experiments were repeated >=3 times, and representative graphs are presented. Compared with the NC group, *P

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Experimental details: Fig. 1 Responses to Candida spp . in neonatal immune cells. a Phagocytosis of Candida in monocytes (boxes and whiskers), including a representative flow microscopy diagram (white bar = ~10 um). Data pooled from multiple experiments over 14 months (9 to 17 subjects per age group; see Supplemental Data for clinical information on preterm subjects); b IL-1beta and c IL-6 response (blood mononuclear cells) to C. albicans or C. parapsilosis (24 h stimulation; 10 to 18 subjects per age group; boxes and whiskers); ( d ) IL-1beta (24 h; 11 to 21 subjects per age group; boxes and whiskers) and e representative gating for pro-IL-1beta (5 h LPS stimulation), gated on CD14-expressing cells (black = fluorescent-minus one control; orange = unstimulated; blue = LPS; representative preterm sample is from a 26 weeks' infant); f pro-IL-1beta (5 h) or g IL-6 (24 h) in response to LPS, zymosan or curdlan (mononuclear cells; 11 to 21 subjects per age group; boxes and whiskers); for b and c , data was pooled from multiple experiments assayed in four ELISA batches with similar distribution of samples per age group

Supportive validation

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Experimental details: Fig. 5 Gene expression and translation of dectin-1 signaling proteins. a Illustration of selected signaling molecules downstream of dectin-1; b Polysome profiles and c quantification of signalosome genes (qPCR) in monosome, disome, and light and heavy polysome fractions (monocytes). Data are from 4 subjects per age group (boxes and whiskers; RQ = relative quantification); d Quantification of signalosome genes (qPCR) in total RNA fractions (4 to 5 subjects/age group; mean +- SD); e Surface expression of dectin-1 (flow cytometry, mononuclear cells, gated on CD14-expressing cells; data pooled from 10 to 23 subjects per age group; boxes and whiskers); f Representative (cropped) Western blot of MALT1 and Bcl10 protein expression in monocytes after 0 to 60 min LPS stimulation. Representative blot is from a 29 weeks gestation sample. Images cropped from same blot probes with each antibody; cumulative quantification of 4 independent Western blot experiments for g MALT1 and h Bcl10 (mean +- SD)

Supportive validation

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Experimental details: Fig. 8 Inhibition glycolysis results in loss of MALT1 protein expression. Effect of blocking glycolysis (using 2-DG) or of blocking translation (using cycloheximide, as control) on MALT1 protein expression (monocytes). a MALT1 protein was detected by Western blot (left panel; representative of two experiments; cropped images from same blot probed with each antibody) at 8 h and 19 h. Lymphoblastoid cell line (LCL) lysate used as positive control for MALT1 protein expression; MALT1 protein detection ( b ) at 16 h and ( c ) over time (intracellular staining by flow cytometry, gated on CD14-expressing cells; MFI mean fluorescence intensity; dotted line: signal for fluorescence-minus-one staining control MFI level; boxes and whiskers with a paired 2-sided t -test in b ; mean +- SD in c and d ; d corresponding cell viability over time (mean +- SD); 6 subjects. e Effect of MALT1 inhibition on IL-1beta, IL-6, and lactate production at rest and following LPS (mononuclear cells; boxes and whiskers with 2-sided paired t -tests); f correlation between LPS-induced IL-1beta and IL-6, and lactate production (Spearman' r ; * p < 0.05; with dotted regression line); 8 subjects. All experiments were conducted in adult cells

Supportive validation

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Experimental details: Increased proportion of non-classical blood monocytes with higher inducible co-stimulator ligand (ICOS-L) expression in sarcoidosis patients. (a) Representative fluorescence activated cell sorter (FACS) dot-plot gated on blood monocyte populations in peripheral blood mononuclear cells (PBMCs) showing the frequencies of classical, intermediate and non-classical (proinflammatory) monocytes in sarcoidosis patients ( n = 11) and healthy controls ( n = 13). (b) Box-plots represent the % CD14 high CD16 low monocytes (classical) in sarcoidosis patients and healthy controls (upper left) and the intensities of ICOS-L [as mean fluorescence intensity (MFI)] on classical monocytes in patients and controls (upper right). Box-plots represent the % CD14 low CD16 high monocytes (non-classical) in sarcoidosis patients and healthy controls (lower left) and the intensities of ICOS-L (as MFI) on non-classical monocytes in patients and controls (lower right). P -values were calculated using the Mann-Whitney U -test.

Supportive validation

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Experimental details: Fig. 5 Characterization of EP2-positive immune cells. a - d Peripheral whole blood (at least 5 subjects per group) was stained with anti-CD14, anti-CD16, anti-EP2, anti-TLR2, anti-HLA-DR, anti-CD11b or anti-TLR4. The percentages of different phenotypes of EP2 + or EP2 - monocytes and neutrophils were determined by flow cytometry. e PBMCs isolated from at least 3 subjects per group were stimulated with LPS (100 ng/ml) for 72 h. Cytokine production was measured by flow cytometry. f Peripheral whole blood from 7 subjects per group was incubated with E. coli for 30 min. ROS production in neutrophils and monocytes was assessed by flow cytometry. The data are expressed as the mean +- SEM of individual subjects or plots of individual data. The error bar represented SEM and the horizontal line represented the median. Statistical analyses were analyzed using the Wilcoxon signed-rank test. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not statistically significant

Supportive validation

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Experimental details: Figure 1. Characterization of hUC-MSCs. (A) Morphological observations of hUC-MSCs. Umbilical cord tissues were cultured for >15 days and long spindle-shaded fibroblastic cells were observed around the tissue using Zeiss light microscopy (scale bar, 100 um). (B) Phenotyping of hUC-MSCs. hUC-MSCs were stained with a fluorescein-labeled antibodies (CD34, CD45, CD73, CD90, CD105, CD14, CD19 and HLA-DR) and analyzed with a flow cytometer. (C) Adipogenic and (D) osteogenic differentiation of hUC-MSCs. hUC-MSCs were cultured in adipogenic and osteogenic medium, respectively. Lipid droplets in the adipocytes are presented with Oil Red O staining (scale bar, 100 um). hUC-MSCs-derived osteoblasts were detected with Alizarin Red staining (scale bar, 200 um). (E) hUC-MSCs inhibit the proliferation of CFSE-labeled CD4 + T cells, which were activated by Con A stimulation. Experiments were repeated three times and representative graphs and images are presented. hUC-MSC, human umbilical cord-derived mesenchymal stem cell; MSC Sup, culture supernatant of hUC-MSCs; Con A, concanavalin A; CFSE, carboxyfluorescein succinimidyl ester.

Supportive validation

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Experimental details: Figure 1. Upregulation of NLRP3 inflammasome mRNA levels in PBMCs in patients with SAP and AMI, compared with non-CHD controls. (A) PBMCs were isolated from peripheral blood and the positive rate of CD14 was calculated to be >=95% of PBMCs by flow cytometry. RT-PCR assays were performed to quantify the mRNA levels of (B) NLRP3, (C) ASC and (D) caspase-1 in PBMCs of each group. *P

Supportive validation

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Experimental details: Figure 3 Generation of patient specific iPSC-derived macrophages. Patient iPSCs have been differentiated into macrophages and compared to macrophages from a healthy iPSC line (hCD34_iPSC16). ( A ) Microscopic analysis of patient iPSCs in cytospin images after Pappenheim staining (left, scale bar = 20 um) or in brightfield images (middle scale bar 200 um, right scale bar = 100 um). ( B ) Representative flow cytometric analysis of CD11b, CD14, CD163 and CD45 expression on patient iPSCs and healthy macrophages of two independent experiments. Blue: Isotype. Pink: Surface marker. FC = fold change of the median fluorescent intensity. ( C ) Flow cytometric analysis of IFN-gammaR1 (top) and IFN-gammaR2 (bottom) expression on healthy and patient iPSC-derived macrophages. Blue: Isotype. Pink: Surface marker. Expression has been quantified by plotting the difference of the median fluorescent intensity (DeltaMFI). Each dot represents macrophages from an independent harvest and from at least three independent differentiations ( n = 4-7, mean +- SD, Kruskal-Wallis with Dunn''s multiple comparison). Red line shows DeltaMFI of 0. ( D ) GM-CSF clearance of healthy and patient iPSC-derived macrophages over a time of 48 h. Concentrations have been normalized to control well containing no cells (media only) ( n = 3, mean +- SD; each dot represents macrophages from an independent harvest and from at least three independent differentiations). ( E ) Representative flow cytometric (top) and microsco

Supportive validation

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Experimental details: Figure 1. CASC15 -KO promotes the differentiation of acute myeloid leukemia cells. (A) Apoptosis in CASC15 -KO and empty vector-transduced (control) OCI-AML5 cell lines after 24 h of depletion of granulocyte-macrophage colony-stimulating factor (annexin-FITC/Sytox blue flow cytometry). (B) Expression of SOX4 during in vitro differentiation of CASC15 -KO and control OCI-AML5 cell lines. All cells were treated with 0.1 mM all- trans retinoic acid (ATRA) and 1 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) over 72 h in three independent experiments. Total RNA was extracted before, after 24 h and after 72 h of treatment, DNase-digested and transcribed to cDNA. A quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR green chemistry with subsequent melting curve analysis in technical triplicates. The 2-ddCt was calculated relative to the pre-determined housekeeping gene encoding succinate dehydrogenase complex subunit C ( SDHC ). (C) Baseline expression of the monocyte/macrophage markers CD11b (integrin subunit alpha M, ITGAM), CD11c (integrin subunit alpha X, ITGAX), and CD14, the granulocyte marker CD15 (fucosyltransferase 4, FUT4), and the general myeloid marker CD13 (aminopeptidase N, APN) in CASC15 -KO and control cells. The percentages of positive cells, quantified by flow cytometry after 72 h, are shown. (D-F) Growth rate and CD11c myeloid cell surface marker expression of CASC15 and control cell lines during drug-induced in vitro differentiation

Supportive validation

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Experimental details: Fig 6 Human neutrophils incubated with supernatants from T . cruzi -infected HFF cells have enhanced expression of CD11b. Flow cytometry analysis of live neutrophils (CD16 + CD66b + CD14 - HLA-DR - ) incubated for 16 hours with media only (A), supernatants from uninfected HFF cells (B), or supernatants of HFF cells infected for 4 days with T . cruzi CL-14 (C) or CL Brener (D). Percentage of cells expressing high levels of the activation marker CD11b (E) and mean fluorescent intensity (MFI) of CD11b (F). * p < 0.05, ** p < 0.01 (one-way ANOVA with Tukey''s post-test comparing the indicated treatments). (A-D) Images are representative of three independent experiments. (E-F) Data from three independent experiments (mean and s.e.m. ).

Supportive validation

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Experimental details: Figure 2 Flow cytometry of CD cell surface markers for cells cultured under hypoxia and normoxia. The positive CD markers for MSCs as detected by the fluorescent antibodies anti-CD73 FITC, anti-CD105 PE, and anti-CD90 PE Cy7. The negative markers of MSCs were detected using anti-CD14 FITC, anti-CD45 PerCP, anti-CD34-R-PE, and anti-CD19 PE-Cy7 antibodies. Unstained cell for each condition was used as negative controls.