Journal: Journal of Neuroinflammation

Article Title: Glimepiride reduces CD14 expression and cytokine secretion from macrophages

doi: 10.1186/1742-2094-11-115

Figure Lengend Snippet: Glimepiride releases CD14 from RAW 264 cells. (A) The amounts of CD14 in RAW 264 cells treated for one hour with control medium (□) or glimepiride (■) as shown. Values are means ± SD, from triplicate experiments performed 4 times, n = 12. (B) The amounts of CD14 in supernatants from RAW 264 cells treated for one hour with control medium (□) or glimepiride as shown (■). Values are means ± SD, from triplicate experiments performed 4 times, n = 12. (C) Immunoblots showing the amounts of CD14, PrP C , CD55 and caveolin in extracts from RAW 264 cells treated for 1 hour with control medium (i) or 5 μM glimepiride (ii). (D) The amounts of CD14 in cells (□) or supernatants (■) from microglial cells treated for 1 hour with control medium, 5 μM glimepiride or 5 μM glipizide. Values are mean units CD14 ± SD, from triplicate experiments performed 3 times, n = 9. *Cellular CD14 significantly less than control cells. **supernatant CD14 significantly greater than control supernatants. (E) Blot showing the amounts of CD14 in supernatants from microglial cells treated with concentrations of glimepiride as shown for one hour.

Article Snippet: Cells were pre-treated with test compounds including glimepiride, glipizide, p-chloromercuriphenylsulphonate (p-CMPS), polymyxin B or phosphatidylinositol (PI)-PLC derived from Bacillus cereus, (all obtained from Sigma) or with antibodies to CD14 (goat polyclonal IgG anti-mouse CD14 (R&D Systems, http://www.rndsystems.com ), PrP C (mouse mAb 4 F2) or CD55 (rat mAb 3D5 Abcam) for one hour before the addition of LPS (Sigma, http://www.sigmaaldrich.com ), PrP82-146, αSN or Aβ 42 .

Techniques: Control, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Glimepiride reduces CD14 expression and cytokine secretion from macrophages

doi: 10.1186/1742-2094-11-115

Figure Lengend Snippet: Glimepiride reduces cytokine secretion from RAW 264 cells incubated with PrP82-146. (A) The concentrations of TNF produced by RAW 264 cells incubated with PrP82-146 (●), PrP82-146scrambled (■) or PrP82-146 and 1 μg/ml polymyxin B (□). Values are means ± SD from triplicate experiments performed 4 times, n = 12. (B) The concentrations of TNF produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (□) and incubated with PrP82-146. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (C) There was a significant correlation between the amounts of cellular CD14 of RAW 264 cells treated with glimepiride (0.3 to 5 μM) for 1 hour and TNF production after the addition of 50 μM PrP82-146, Pearson’s coefficient = 0.858, P < 0.01. (D) The concentrations of IL-1β produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (□) and incubated with PrP82-146. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (E) The concentrations of IL-6 produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (□) and incubated with PrP82-146. Values are means ± SD from triplicate experiments performed 4 times, n = 12.

Article Snippet: Cells were pre-treated with test compounds including glimepiride, glipizide, p-chloromercuriphenylsulphonate (p-CMPS), polymyxin B or phosphatidylinositol (PI)-PLC derived from Bacillus cereus, (all obtained from Sigma) or with antibodies to CD14 (goat polyclonal IgG anti-mouse CD14 (R&D Systems, http://www.rndsystems.com ), PrP C (mouse mAb 4 F2) or CD55 (rat mAb 3D5 Abcam) for one hour before the addition of LPS (Sigma, http://www.sigmaaldrich.com ), PrP82-146, αSN or Aβ 42 .

Techniques: Incubation, Produced, Control

Journal: Journal of Neuroinflammation

Article Title: Glimepiride reduces CD14 expression and cytokine secretion from macrophages

doi: 10.1186/1742-2094-11-115

Figure Lengend Snippet: RAW264 and microglial cells recover from glimepiride-induced suppression of cytokine production. (A) The amounts of CD14 in RAW 264 cells treated with control medium (□) or 5 μM glimepiride for time periods as shown (■). Values are means ± SD from triplicate experiments performed twice, n = 6. (B) The concentrations of TNF produced by RAW 264 cells pre-treated with control medium (□) or 5 μM glimepiride for time periods as shown (■) and incubated with 50 μM PrP82-146 for 24 hours. Values are means ± SD from triplicate experiments performed twice, n = 6. (C) The amounts of CD14 in microglial cells treated with control medium (□) or 5 μM glimepiride for time periods as shown (■). Values are means ± SD from triplicate experiments performed twice, n = 6. (D) The concentrations of TNF produced by microglial cells pre-treated with control medium (□) or 5 μM glimepiride for time periods as shown (■) and incubated with 50 μM PrP82-146 for 24 hours. Values are means ± SD from triplicate experiments performed twice, n = 6.

Article Snippet: Cells were pre-treated with test compounds including glimepiride, glipizide, p-chloromercuriphenylsulphonate (p-CMPS), polymyxin B or phosphatidylinositol (PI)-PLC derived from Bacillus cereus, (all obtained from Sigma) or with antibodies to CD14 (goat polyclonal IgG anti-mouse CD14 (R&D Systems, http://www.rndsystems.com ), PrP C (mouse mAb 4 F2) or CD55 (rat mAb 3D5 Abcam) for one hour before the addition of LPS (Sigma, http://www.sigmaaldrich.com ), PrP82-146, αSN or Aβ 42 .

Techniques: Control, Produced, Incubation

Journal: Journal of Neuroinflammation

Article Title: Glimepiride reduces CD14 expression and cytokine secretion from macrophages

doi: 10.1186/1742-2094-11-115

Figure Lengend Snippet: Glimepiride reduces lipopolysaccharide (LPS)-induced cytokine secretion. (A) The concentrations of TNF produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (■) and incubated with LPS. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (B) The concentrations of IL-1β produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (■) and incubated with LPS. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (C) The concentrations of IL-6 produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (■) and incubated with LPS. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (D) The concentrations of TNF produced by microglial cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (■) and incubated with LPS. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (E) The concentrations of TNF produced by RAW 264 cells pre-treated with control medium (□) or glimepiride as shown (■) and incubated with 10 ng/ml LPS. Values are means ± SD from triplicate experiments performed 3 times, n = 9. (F) There was a significant correlation between the CD14 content of RAW 264 cells treated with glimepiride (0.3 to 5 μM) and the concentrations of TNF produced in response to 10 ng/ml LPS, Pearson’s coefficient = 0.886.

Article Snippet: Cells were pre-treated with test compounds including glimepiride, glipizide, p-chloromercuriphenylsulphonate (p-CMPS), polymyxin B or phosphatidylinositol (PI)-PLC derived from Bacillus cereus, (all obtained from Sigma) or with antibodies to CD14 (goat polyclonal IgG anti-mouse CD14 (R&D Systems, http://www.rndsystems.com ), PrP C (mouse mAb 4 F2) or CD55 (rat mAb 3D5 Abcam) for one hour before the addition of LPS (Sigma, http://www.sigmaaldrich.com ), PrP82-146, αSN or Aβ 42 .

Techniques: Produced, Control, Incubation

Journal: Journal of Neuroinflammation

Article Title: Glimepiride reduces CD14 expression and cytokine secretion from macrophages

doi: 10.1186/1742-2094-11-115

Figure Lengend Snippet: Glimepiride induced immunosuppression is reversed by pCMPS. (A) The amounts of CD14 in RAW 264 cells treated for 1 hour with control medium or 5 μM glimepiride in the presence of control medium (□) or 200 μM pCMPS (■). Values are means ± SD from triplicate experiments performed 4 times, n = 12. *CD14 significantly greater than those of cells treated with glimepiride. (B) The concentrations of TNF produced by RAW 264 cells pre-treated with control medium (□), 5 μM glimepiride (■), or 5 μM glimepiride and 200 μM pCMPS (striped bars) and incubated with 50 μM PrP82-146, 50 μM Aβ 1–42 or 50 μM αSN. Values are means ± SD from triplicate experiments performed 4 times, n = 12. *TNF significantly less than control cells incubated with peptides. (C) The concentrations of TNF produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glimepiride and 200 μM pCMPS ( ■ ) and incubated with LPS as shown. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (D) The concentrations of TNF produced by microglial cells pre-treated with control medium (□), 5 μM glimepiride (■), or 5 μM glimepiride and 200 μM pCMPS (striped bars) and incubated with 50 μM PrP82-146 or 50 μM Aβ 1–42 . Values are means ± SD from triplicate experiments performed twice, n = 6. *TNF significantly less than control cells incubated with peptides.

Article Snippet: Cells were pre-treated with test compounds including glimepiride, glipizide, p-chloromercuriphenylsulphonate (p-CMPS), polymyxin B or phosphatidylinositol (PI)-PLC derived from Bacillus cereus, (all obtained from Sigma) or with antibodies to CD14 (goat polyclonal IgG anti-mouse CD14 (R&D Systems, http://www.rndsystems.com ), PrP C (mouse mAb 4 F2) or CD55 (rat mAb 3D5 Abcam) for one hour before the addition of LPS (Sigma, http://www.sigmaaldrich.com ), PrP82-146, αSN or Aβ 42 .

Techniques: Control, Produced, Incubation

Journal: Journal of Neuroinflammation

Article Title: Glimepiride reduces CD14 expression and cytokine secretion from macrophages

doi: 10.1186/1742-2094-11-115

Figure Lengend Snippet: CD14 mediates cytokine secretion from peptide stimulated RAW 264 cells. (A) The concentrations of TNF produced by RAW 264 cells pre-treated with antiserum to CD14 (■), PrP C (□) or CD55 (striped bars) and incubated with 50 μM PrP82-146, Aβ 1–42 or αSN. Values are means ± SD from triplicate experiments performed 3 times, n = 9. *TNF significantly less than those of control cells incubated with peptides. (B) The concentrations of TNF produced by microglial cells derived from CD14 wild type (□) or CD14 knockout (■) mice incubated with PrP82-146 or Aβ 1–42 . Values are means ± SD from triplicate experiments performed 3 times, n = 9. *TNF significantly less than those from CD14 wild type cells incubated with peptides.

Article Snippet: Cells were pre-treated with test compounds including glimepiride, glipizide, p-chloromercuriphenylsulphonate (p-CMPS), polymyxin B or phosphatidylinositol (PI)-PLC derived from Bacillus cereus, (all obtained from Sigma) or with antibodies to CD14 (goat polyclonal IgG anti-mouse CD14 (R&D Systems, http://www.rndsystems.com ), PrP C (mouse mAb 4 F2) or CD55 (rat mAb 3D5 Abcam) for one hour before the addition of LPS (Sigma, http://www.sigmaaldrich.com ), PrP82-146, αSN or Aβ 42 .

Techniques: Produced, Incubation, Control, Derivative Assay, Knock-Out

Journal: Cancer Research

Article Title: Resveratrol Inhibits Myeloma Cell Growth, Prevents Osteoclast Formation, and Promotes Osteoblast Differentiation

doi: 10.1158/0008-5472.can-05-0651

Figure Lengend Snippet: Figure 5. Resveratrol impairs RANK and NFATc1 expression as well as NF-nB translocation while up-regulating c-fms, CD14, and CD11a expression. A, relative gene expression of NFATc1 measured by quantitative real-time PCR in monocytes cultured for 3 or 7 days with M-CSF or M-CSF + RANKL in the absence or presence of 100 Amol/L resveratrol (left) or in 7-day cultures treated with M-CSF or M-CSF + RANKL in the presence of increasing concentrations of resveratrol (right). Levels of mRNA expression were normalized to h-actin and are represented as induction fold compared with cells cultured with M-CSF alone for 3 days (left) and 7 days (right). Columns, mean of four cultures; bars, SD. *, P < 0.001. B, left, relative gene expression of RANK in monocytes cultured for 3 or 7 days with M-CSF or M-CSF + RANKL in the absence or presence of 100 Amol/L resveratrol. Levels of mRNA expression were normalized to h-actin and are represented as induction fold compared with cells cultured with M-CSF alone at day 3. Columns, mean of four cultures; bars, SD. *, P < 0.001. Right, level of RANK expression on the cell surface analyzed by flow cytometry. Monocytes were cultured for 3 days with M-CSF + RANKL in the absence or presence of 100 Amol/L resveratrol. Thin line, isotypic control; filled histogram, untreated cells; thick line, cells treated with resveratrol. C, detection by immunofluorescence of RANKL-induced NF-nB nuclear translocation in cultures of monocytes treated with 100 Amol/L resveratrol for 1 hour or 3 days. In absence of RANKL, most of the cells showed a cytoplasmic localization of NF-nB (W/O), whereas after 30 minutes RANKL exposure f20% cells displayed a strong nuclear staining (With RANKL). Asterisks, cell nuclei. Graphs show the number of cells with NF-nB nuclear translocation in percentage of the total number of scrutinized cells (counting >400 cells per culture). Columns, mean of four cultures; bars, SD. *, P < 0.01. D, relative gene expression of c-fms, CD14, and CD11a in cells cultured for 3 or 7 days with M-CSF or M-CSF + RANKL in the absence or presence of 100 Amol/L resveratrol. Levels of mRNA expression were normalized to h-actin and are represented as induction fold compared with cells cultured with M-CSF alone at day 3. Columns, mean of four cultures; bars, SD. *, P < 0.001.

Article Snippet: Briefly, PBMCs resuspended in 2% FCS in PBS were incubated first with biotinylated anti-human CD14 goat antibody (R&D Systems, Abingdon, United Kingdom) for 15 minutes at 4jC and then with Magcellect streptavidin ferrofluid (R&D Systems) for 15minutes at 4jC.

Techniques: Expressing, Translocation Assay, Gene Expression, Real-time Polymerase Chain Reaction, Cell Culture, Flow Cytometry, Control, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients

doi: 10.1371/journal.pone.0051867

Figure Lengend Snippet: A–D. Immunocytochemistry showed distinct phagocytosis of polystyrene beads (green) by monocytes treated with 31.25 µg/ml glatiramer acetate (GA) for 24 h (red, labelled with anti-CD14 antibody) as compared to medium, human serum albumin (HA), and mannitol (Mt) vehicle control. Scale bar: 50 µm. E. Representative z-stack series of confocal microscopic images demonstrate that the particles were completely internalised and not merely attached to the outer membrane. Scale bar: 10 µm.

Article Snippet: After 30 minutes incubation with fluorescent latex beads (FluoresbriteTM YG carboxylate microspheres; 1 μm diameter; Polysciences, Eppelheim, Germany) at 37°C, cover slips were washed three times and cells were stained with the CD14 antibody (primary: rabbit anti-human IgG, Abcam, Cambridge, UK; secondary: goat anti-rabbit IgG, AbD Serotec, Düsseldorf, Germany) at 1∶100 dilution in DMEM+.

Techniques: Immunocytochemistry, Control, Membrane

Journal: PLoS ONE

Article Title: Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients

doi: 10.1371/journal.pone.0051867

Figure Lengend Snippet: A–B. Gated monocytes in a forward vs. side scatter dot-plot analysis of peripheral blood mononuclear cells. Monocytes were then gated according to their surface expression of CD14 and CD16. Flow cytometric analysis of phagocytosis revealed that CD14 ++ CD16 + monocytes (gate III) engulfed polystyrene beads more effectively than the other subsets (gate I and II). CI–III. The mean fluorescence intensities represent the amount of incorporated fluorescent latex particles phagocytosed by 3×10 5 cells. D. Increase in the percentage of CD14 + CD16 + monocytes after 4 h and 8 h of treatment with 31.25 µg/ml glatiramer acetate (GA) in MACS isolated monocytes. Data are expressed as mean percentages ± SEM of three independent experiments. Significant effects vs. controls are indicated by asterisks (*p<0.05, **p<0.01, and ***p<0.001 using Bonferroni's Multiple Comparison Test) as determined by one-way ANOVA. E. Slight but not significant increase of CD16 expression after GA treatment.

Article Snippet: After 30 minutes incubation with fluorescent latex beads (FluoresbriteTM YG carboxylate microspheres; 1 μm diameter; Polysciences, Eppelheim, Germany) at 37°C, cover slips were washed three times and cells were stained with the CD14 antibody (primary: rabbit anti-human IgG, Abcam, Cambridge, UK; secondary: goat anti-rabbit IgG, AbD Serotec, Düsseldorf, Germany) at 1∶100 dilution in DMEM+.

Techniques: Expressing, Fluorescence, Isolation, Comparison

Journal: PLoS ONE

Article Title: Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients

doi: 10.1371/journal.pone.0051867

Figure Lengend Snippet: Mean fluorescence intensities of CD36, CD210, and TIM-3 (± SEM) of three independent experiments after a treatment period of 24 h according to the different monocyte subsets, i.e. CD14 + CD16 − and CD14 + CD16 + monocytes.

Article Snippet: After 30 minutes incubation with fluorescent latex beads (FluoresbriteTM YG carboxylate microspheres; 1 μm diameter; Polysciences, Eppelheim, Germany) at 37°C, cover slips were washed three times and cells were stained with the CD14 antibody (primary: rabbit anti-human IgG, Abcam, Cambridge, UK; secondary: goat anti-rabbit IgG, AbD Serotec, Düsseldorf, Germany) at 1∶100 dilution in DMEM+.

Techniques: Fluorescence

Journal: PLoS ONE

Article Title: Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients

doi: 10.1371/journal.pone.0051867

Figure Lengend Snippet: The MFI displays the amount of incorporated beads phagocytosed by 3×10 5 cells. Data are expressed as means of the mean fluorescence intensities (MFI) ± SEM of three independent experiments. Pre-treatment of monocytes with anti-CD14, anti-CD16, anti-CD32, anti-CD210, and anti-TIM3 antibodies reduced glatiramer acetate (GA) induced phagocytosis of polystyrene beads. The anti-CD36 and, at the highest concentration, the anti-IL10 antibody most effectively suppressed phagocytosis. Significant effects vs. GA 31.25 µg/ml are indicated by asterisks (*p<0.05, **p<0.01, and ***p<0.001 using Bonferroni's Multiple Comparison Test) as determined by one-way ANOVA. B–C. The anti-CD36 antibodies of two different clones show a diverse impact on the phagocytosis of polystyrene beads by untreated cells, whereas both antibodies effectively decreased the GA induced phagocytosis.

Article Snippet: After 30 minutes incubation with fluorescent latex beads (FluoresbriteTM YG carboxylate microspheres; 1 μm diameter; Polysciences, Eppelheim, Germany) at 37°C, cover slips were washed three times and cells were stained with the CD14 antibody (primary: rabbit anti-human IgG, Abcam, Cambridge, UK; secondary: goat anti-rabbit IgG, AbD Serotec, Düsseldorf, Germany) at 1∶100 dilution in DMEM+.

Techniques: Fluorescence, Concentration Assay, Comparison, Clone Assay

Journal:

Article Title: Development and prevention of advanced diabetic nephropathy in RAGE-overexpressing mice

doi:

Figure Lengend Snippet: Generation and characterization of RAGE transgenic mice. (a) Transgene construct. The human RAGE genomic sequence, beginning with the initiator codon in exon 1 and ending 129 bp downstream from the last exon, was placed under the transcriptional control of the murine flk-1 promoter. The flk-1 fragment encompassed the 5′ untranslated region (dark gray box), sharing the ATG codon with the RAGE fragment. (b) Transgene-derived transcripts. Total RNAs isolated from various tissues of line 102 and 103 heterozygotes were analyzed by RT-PCR. The granulation tissue — the focus of angiogenesis — was prepared by punching out an area of the dorsal skin about 4–5 mm in diameter. The PCR-amplified products had a chain length of 354 bp as predicted and were sequence-verified. (c) Immunofluorescence staining of kidneys from line 102 RAGETg or nontransgenic control at 4 months of age using anti-human RAGE-specific polyclonal Ab. Original magnification, ×430. (d) Translation products of the transgene. Extracts of isolated ECs from renal cortex or peripheral blood monocytes of line 102 RAGETg or the nontransgenic control were immunodetected with the human RAGE Ab. Specific bands were marked at 55 kDa in line 102 RAGETg EC and monocyte extracts. VE-cad, VE-cadherin, i.e., an EC marker; CD14, a monocyte marker.

Article Snippet: Membranes were blocked with 5% nonfat dry milk in PBS and 0.1% Tween-20, and then incubated with 1:100 dilution of goat anti–vascular endothelial-cadherin (anti–VE-cadherin) polyclonal Ab (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA), 1:100 dilution of goat anti-mouse CD14 polyclonal Ab (Santa Cruz Biotechnology Inc.), or 0.25 μg/ml of immunoaffinity-purified anti-RAGE polyclonal Ab raised against 16-amino-acid synthetic peptides (QRRQRRGEERKAPENQ) that corresponded to amino acid residues 364–379 of the human RAGE protein ( 11 ).

Techniques: Transgenic Assay, Construct, Sequencing, Control, Derivative Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Amplification, Immunofluorescence, Staining, Marker

Journal: European journal of immunology

Article Title: Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns.

doi: 10.1002/eji.200425995

Figure Lengend Snippet: Figure 1. 1,25(OH)2D3 down-regulates TLR2 and TLR4 in a time- and dose-dependent manner. (A) Monocytes were cultured in presence or absence of 10–7 M 1,25(OH)2D3 in a time course from 12, 24, 48 to 72 h. Positive percentages of cells expressing TLR2, TLR4 and CD14 were determined by flow cytometry. Changes over time were calculated as compared to TLR expression of untreated cells, which was set to 1. Data represent means SD; *p<0.05, ***p<0.001. (B) Down-regulation of TLR mRNA after incubation with 1,25(OH)2D3. Quantitative RT-PCR for human TLR2 and TLR4 was conducted with human monocytes and normalized to b-actin gene expression. Cells were incubated with 10–7 M 1,25(OH)2D3 for 3, 6 and 9 h or with medium alone. Data show results from four independent experiments and are expressed as fold changes. Error bars show means SD; statistically significant changes of 1,25(OH)2D3-treated monocytes vs. untreated cells: *p<0.05, **p<0.01 as calculated by one-way ANOVA. (C) Effect of 1,25(OH)2D3 on TLR2, TLR4 and CD14 expression is dose-dependent. Human monocytes were incubated in absence or in presence of increasing amounts of 1,25(OH)2D3 ranging from 10–11–10–7 M for 48 h. The MFI of monocyte TLR expression was measured by flow cytometry. Data from four individual experiments represent means SD; *p<0.05, **p<0.01 vs. medium alone. (D) Representative two-dimensional dot plot histograms of one from three individually analyzed apparently healthy volunteers show both reduced TLR and increased CD14 expression on monocytes after single administration of 10–7 M 1,25(OH)2D3 which peaked after 72 h. We further observed an increase of TLR4 expression in untreated cells after 72 h.

Article Snippet: Cells were washed twice with staining buffer and the following phospho-specific mAb were added for 30 min at room temperature: PE-p38 (pT180/pY182, mouse IgG1, clone 36), PE-ERK1/2 (pT202/pY204, mouse IgG1), both BD Pharmingen, and CD14-FITC (mouse IgG2a, clone UCH-M1; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Cell Culture, Expressing, Flow Cytometry, Incubation, Quantitative RT-PCR, Gene Expression

Journal: European journal of immunology

Article Title: Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns.

doi: 10.1002/eji.200425995

Figure Lengend Snippet: Figure 2. (A) Reduced SAPK phosphorylation upon TLR-ligand engagement in monocytes treated with 1,25(OH)2D3. Nega- tively depleted CD14+ cells pre-incubated with or without 1,25(OH)2D3 were stimulated for 10 min with either 10 ng LPS or 10 lg LTA. Cells were fixed, permeabilized and phospho- specific mAb were added to determine p38 and p42/44 activation. Colored histograms show cells treated with med- ium only while open histograms show activated cells stimu- lated with LPS or LTA. Incubation of 1,25(OH)2D3 prior to TLR engagement (10–7 M, shaded histograms) resulted in reduced phosphorylation of p38 and ERK1/2. Histograms shown are representative of three individually performed experiments. (B, C) Influence of 1,25(OH)2D3 on cell viability. Monocytes were treated with or without 10–7 M 1,25(OH)2D3, and annexin V- FITC/PI staining was performed at each time point (n=3). No difference was observed between untreated cells and cells that were incubated with 1,25(OH)2D3. Representative dot plot diagram depicted shows the impact of 1,25(OH)2D3 on monocytes viability after 48 h.

Article Snippet: Cells were washed twice with staining buffer and the following phospho-specific mAb were added for 30 min at room temperature: PE-p38 (pT180/pY182, mouse IgG1, clone 36), PE-ERK1/2 (pT202/pY204, mouse IgG1), both BD Pharmingen, and CD14-FITC (mouse IgG2a, clone UCH-M1; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Phospho-proteomics, Incubation, Activation Assay, Staining

Journal: European journal of immunology

Article Title: Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns.

doi: 10.1002/eji.200425995

Figure Lengend Snippet: Figure 3. (A) Decreased intracellular TNF-a production in 1,25(OH)2D3-treated CD14+ cells upon TLR ligand induction. Monocytes were incubated for 48 h in absence or presence of various 1,25(OH)2D3 concentrations ranging from 10–7–10–11 M and afterwards stimulated for 4 h with 10 ng/mL LPS or 10 lg/mL LTA, respectively. Cells were double-stained with CD14-FITC, TNF-a-PE or with corresponding isotype antibodies and analyzed by flow cytometry. Two-color dot plots are from one representative experiment of three. Values in gate refer to the percentage of TNF-a+CD14+ cells. (B) TLR ligand triggered TNF-a secretion in the presence or absence of vitamin D3 in supernatants. Negatively selected monocytes were incubated with 1,25(OH)2D3, and stimulated with either 10 ng/mL LPS or 10 lg/mL LTA for 4 h. Supernatants were collected and analyzed for TNF-a concentrations by chemiluminescence. (C) Diminished TNF-a expression in monocytes pre-incubated with either anti-TLR2 or anti-TLR4 antibodies. Human monocytes (1106/mL) were treated with either 10 ng/mL LPS or 10 lg/mL LTA for 4 h. Cells in the anti-TLR4- or anti-TLR2- treated sample were pre-incubated with 20 lg/mL HTA125 or 20 lg/mL TL2.1 for 1 h prior to addition of LPS or LTA. Intracellular TNF-a production (MFI) was measured as described in Materials and methods. Both clones HTA125 (TLR4) and TL2.1 (TLR2) substantially inhibited TNF-a expression. Bars show means SD of three independent experiments. Data represent the means SD (n=3); *p<0.05, **p<0.01 vs. control as determined by Tukey-Kramer.

Article Snippet: Cells were washed twice with staining buffer and the following phospho-specific mAb were added for 30 min at room temperature: PE-p38 (pT180/pY182, mouse IgG1, clone 36), PE-ERK1/2 (pT202/pY204, mouse IgG1), both BD Pharmingen, and CD14-FITC (mouse IgG2a, clone UCH-M1; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Incubation, Staining, Flow Cytometry, Expressing, Clone Assay, Control

Journal: European journal of immunology

Article Title: Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns.

doi: 10.1002/eji.200425995

Figure Lengend Snippet: Figure 4. (A) Counteracting effects of 1,25(OH)2D3 on up- regulation of TF by LPS and LTA in phagocytes. Monocytes were incubated with increasing concentrations of 1,25(OH)2D3 and afterwards stimulated with 10 ng LPS or 10 lg LTA for 4 h. Levels of TF on CD14+ cells were measured by flow cytometry. Data from three independent experiments represent the means SD; *p<0.05, **p<0.01 vs. control as determined by Tukey-Kramer. (B) Reduced TF activity on human monocytes activated with 10 ng/mL E. coli LPS for 4 h. Prior to stimulation, cells were cultured for 48 h in absence or presence of various concentrations of 1,25(OH)2D3. Total TF activity of cell lysates was quantified using a standard curve generated with recombinant human TF. Results from five independent experiments represent the means SD; **p<0.01 vs. cells stimulated with LPS alone.

Article Snippet: Cells were washed twice with staining buffer and the following phospho-specific mAb were added for 30 min at room temperature: PE-p38 (pT180/pY182, mouse IgG1, clone 36), PE-ERK1/2 (pT202/pY204, mouse IgG1), both BD Pharmingen, and CD14-FITC (mouse IgG2a, clone UCH-M1; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Incubation, Flow Cytometry, Control, Activity Assay, Cell Culture, Generated, Recombinant

Journal: European journal of immunology

Article Title: Vitamin D3 down-regulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns.

doi: 10.1002/eji.200425995

Figure Lengend Snippet: Figure 5. (A) Diminished RelA translocation in 1,25(OH)2D3- treated monocytes after TLR challenge. Negatively selected CD14+ monocytes were incubated for 48 h in absence or presence of 10–7 M 1,25(OH)2D3 and stimulated with either 10 ng/mL LPS or 10 lg/mL LTA for 30 min. Cells were attached onto microscope slides, fixed and NF-jB/RelA (p65) and PI immunofluorescence staining was performed. Individual and merged pictures out of two independent experiments are shown. No background staining was observed in slides incubated with corresponding isotype antibodies alone (not shown). Original magnifications, 100. (B, C) TLR down- regulation is blocked by the VDR antagonist ZK 159222. Two- and one-dimensional overlay plots of two separately per- formed experiments are presented. While 1,25(OH)2D3-treated monocytes (red cells) show divergent expression levels of TLR and CD14, cells incubated with both ZK 159222 and 1,25(OH)2D3 (orange) and untreated cells (green) are similar in CD14 and TLR expression. Concentrations for 1,25(OH)2D3 and ZK 159222 were 10–7 M and 10–6 M, respectively. ZK 159222 was added 1 h before 1,25(OH)2D3 administration and overall incubation time was 48 h.

Article Snippet: Cells were washed twice with staining buffer and the following phospho-specific mAb were added for 30 min at room temperature: PE-p38 (pT180/pY182, mouse IgG1, clone 36), PE-ERK1/2 (pT202/pY204, mouse IgG1), both BD Pharmingen, and CD14-FITC (mouse IgG2a, clone UCH-M1; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Translocation Assay, Incubation, Microscopy, Immunofluorescence, Staining, Expressing

Journal: Journal of Neuroinflammation

Article Title: Glimepiride reduces CD14 expression and cytokine secretion from macrophages

doi: 10.1186/1742-2094-11-115

Figure Lengend Snippet: Glimepiride releases CD14 from RAW 264 cells. (A) The amounts of CD14 in RAW 264 cells treated for one hour with control medium (□) or glimepiride (■) as shown. Values are means ± SD, from triplicate experiments performed 4 times, n = 12. (B) The amounts of CD14 in supernatants from RAW 264 cells treated for one hour with control medium (□) or glimepiride as shown (■). Values are means ± SD, from triplicate experiments performed 4 times, n = 12. (C) Immunoblots showing the amounts of CD14, PrP C , CD55 and caveolin in extracts from RAW 264 cells treated for 1 hour with control medium (i) or 5 μM glimepiride (ii). (D) The amounts of CD14 in cells (□) or supernatants (■) from microglial cells treated for 1 hour with control medium, 5 μM glimepiride or 5 μM glipizide. Values are mean units CD14 ± SD, from triplicate experiments performed 3 times, n = 9. *Cellular CD14 significantly less than control cells. **supernatant CD14 significantly greater than control supernatants. (E) Blot showing the amounts of CD14 in supernatants from microglial cells treated with concentrations of glimepiride as shown for one hour.

Article Snippet: Samples were applied and the amount of bound CD14 was measured using a goat polyclonal IgG anti-mouse CD14 (R&D Systems), followed by anti-goat IgG conjugated to alkaline phosphatase (Sigma) and 1 mg/ml 4-nitrophenyl phosphate (Sigma).

Techniques: Control, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Glimepiride reduces CD14 expression and cytokine secretion from macrophages

doi: 10.1186/1742-2094-11-115

Figure Lengend Snippet: Glimepiride reduces cytokine secretion from RAW 264 cells incubated with PrP82-146. (A) The concentrations of TNF produced by RAW 264 cells incubated with PrP82-146 (●), PrP82-146scrambled (■) or PrP82-146 and 1 μg/ml polymyxin B (□). Values are means ± SD from triplicate experiments performed 4 times, n = 12. (B) The concentrations of TNF produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (□) and incubated with PrP82-146. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (C) There was a significant correlation between the amounts of cellular CD14 of RAW 264 cells treated with glimepiride (0.3 to 5 μM) for 1 hour and TNF production after the addition of 50 μM PrP82-146, Pearson’s coefficient = 0.858, P < 0.01. (D) The concentrations of IL-1β produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (□) and incubated with PrP82-146. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (E) The concentrations of IL-6 produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (□) and incubated with PrP82-146. Values are means ± SD from triplicate experiments performed 4 times, n = 12.

Article Snippet: Samples were applied and the amount of bound CD14 was measured using a goat polyclonal IgG anti-mouse CD14 (R&D Systems), followed by anti-goat IgG conjugated to alkaline phosphatase (Sigma) and 1 mg/ml 4-nitrophenyl phosphate (Sigma).

Techniques: Incubation, Produced, Control

Journal: Journal of Neuroinflammation

Article Title: Glimepiride reduces CD14 expression and cytokine secretion from macrophages

doi: 10.1186/1742-2094-11-115

Figure Lengend Snippet: RAW264 and microglial cells recover from glimepiride-induced suppression of cytokine production. (A) The amounts of CD14 in RAW 264 cells treated with control medium (□) or 5 μM glimepiride for time periods as shown (■). Values are means ± SD from triplicate experiments performed twice, n = 6. (B) The concentrations of TNF produced by RAW 264 cells pre-treated with control medium (□) or 5 μM glimepiride for time periods as shown (■) and incubated with 50 μM PrP82-146 for 24 hours. Values are means ± SD from triplicate experiments performed twice, n = 6. (C) The amounts of CD14 in microglial cells treated with control medium (□) or 5 μM glimepiride for time periods as shown (■). Values are means ± SD from triplicate experiments performed twice, n = 6. (D) The concentrations of TNF produced by microglial cells pre-treated with control medium (□) or 5 μM glimepiride for time periods as shown (■) and incubated with 50 μM PrP82-146 for 24 hours. Values are means ± SD from triplicate experiments performed twice, n = 6.

Article Snippet: Samples were applied and the amount of bound CD14 was measured using a goat polyclonal IgG anti-mouse CD14 (R&D Systems), followed by anti-goat IgG conjugated to alkaline phosphatase (Sigma) and 1 mg/ml 4-nitrophenyl phosphate (Sigma).

Techniques: Control, Produced, Incubation

Journal: Journal of Neuroinflammation

Article Title: Glimepiride reduces CD14 expression and cytokine secretion from macrophages

doi: 10.1186/1742-2094-11-115

Figure Lengend Snippet: Glimepiride reduces lipopolysaccharide (LPS)-induced cytokine secretion. (A) The concentrations of TNF produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (■) and incubated with LPS. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (B) The concentrations of IL-1β produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (■) and incubated with LPS. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (C) The concentrations of IL-6 produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (■) and incubated with LPS. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (D) The concentrations of TNF produced by microglial cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glipizide (■) and incubated with LPS. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (E) The concentrations of TNF produced by RAW 264 cells pre-treated with control medium (□) or glimepiride as shown (■) and incubated with 10 ng/ml LPS. Values are means ± SD from triplicate experiments performed 3 times, n = 9. (F) There was a significant correlation between the CD14 content of RAW 264 cells treated with glimepiride (0.3 to 5 μM) and the concentrations of TNF produced in response to 10 ng/ml LPS, Pearson’s coefficient = 0.886.

Article Snippet: Samples were applied and the amount of bound CD14 was measured using a goat polyclonal IgG anti-mouse CD14 (R&D Systems), followed by anti-goat IgG conjugated to alkaline phosphatase (Sigma) and 1 mg/ml 4-nitrophenyl phosphate (Sigma).

Techniques: Produced, Control, Incubation

Journal: Journal of Neuroinflammation

Article Title: Glimepiride reduces CD14 expression and cytokine secretion from macrophages

doi: 10.1186/1742-2094-11-115

Figure Lengend Snippet: Glimepiride induced immunosuppression is reversed by pCMPS. (A) The amounts of CD14 in RAW 264 cells treated for 1 hour with control medium or 5 μM glimepiride in the presence of control medium (□) or 200 μM pCMPS (■). Values are means ± SD from triplicate experiments performed 4 times, n = 12. *CD14 significantly greater than those of cells treated with glimepiride. (B) The concentrations of TNF produced by RAW 264 cells pre-treated with control medium (□), 5 μM glimepiride (■), or 5 μM glimepiride and 200 μM pCMPS (striped bars) and incubated with 50 μM PrP82-146, 50 μM Aβ 1–42 or 50 μM αSN. Values are means ± SD from triplicate experiments performed 4 times, n = 12. *TNF significantly less than control cells incubated with peptides. (C) The concentrations of TNF produced by RAW 264 cells pre-treated with control medium (●), 5 μM glimepiride (○) or 5 μM glimepiride and 200 μM pCMPS ( ■ ) and incubated with LPS as shown. Values are means ± SD from triplicate experiments performed 4 times, n = 12. (D) The concentrations of TNF produced by microglial cells pre-treated with control medium (□), 5 μM glimepiride (■), or 5 μM glimepiride and 200 μM pCMPS (striped bars) and incubated with 50 μM PrP82-146 or 50 μM Aβ 1–42 . Values are means ± SD from triplicate experiments performed twice, n = 6. *TNF significantly less than control cells incubated with peptides.

Article Snippet: Samples were applied and the amount of bound CD14 was measured using a goat polyclonal IgG anti-mouse CD14 (R&D Systems), followed by anti-goat IgG conjugated to alkaline phosphatase (Sigma) and 1 mg/ml 4-nitrophenyl phosphate (Sigma).

Techniques: Control, Produced, Incubation

Journal: Journal of Neuroinflammation

Article Title: Glimepiride reduces CD14 expression and cytokine secretion from macrophages

doi: 10.1186/1742-2094-11-115

Figure Lengend Snippet: CD14 mediates cytokine secretion from peptide stimulated RAW 264 cells. (A) The concentrations of TNF produced by RAW 264 cells pre-treated with antiserum to CD14 (■), PrP C (□) or CD55 (striped bars) and incubated with 50 μM PrP82-146, Aβ 1–42 or αSN. Values are means ± SD from triplicate experiments performed 3 times, n = 9. *TNF significantly less than those of control cells incubated with peptides. (B) The concentrations of TNF produced by microglial cells derived from CD14 wild type (□) or CD14 knockout (■) mice incubated with PrP82-146 or Aβ 1–42 . Values are means ± SD from triplicate experiments performed 3 times, n = 9. *TNF significantly less than those from CD14 wild type cells incubated with peptides.

Article Snippet: Samples were applied and the amount of bound CD14 was measured using a goat polyclonal IgG anti-mouse CD14 (R&D Systems), followed by anti-goat IgG conjugated to alkaline phosphatase (Sigma) and 1 mg/ml 4-nitrophenyl phosphate (Sigma).

Techniques: Produced, Incubation, Control, Derivative Assay, Knock-Out

Journal: Journal of Cancer

Article Title: MMP9 and IGFBP1 Regulate Tumor Immune and Drive Tumor Progression in Clear Cell Renal Cell Carcinoma

doi: 10.7150/jca.48664

Figure Lengend Snippet: Validation of hub genes in the high-risk group and low-risk group. (A) ccRCC samples were divided into high monocytes group and low monocytes group based on the expression of CD14. Validation of the mRNA expression of MMP9 (B), F2 (C), HP (D), CXCL13 (E), IGFBP1 (F), VTN (G), VGF (H), MFI2 (I), and SAA1(J) between high-risk group and low-risk group. IHC assay was used to verify the protein expression of MMP9 (K) and IGFBP1 (L) in ccRCC. ccRCC: clear cell renal cell carcinoma. ccRCC: clear cell renal cell carcinoma.

Article Snippet: The primary antibodies and secondary antibodies were as following: MMP9 (1:100, Abclonal, China, A0289); IGFBP1 (1:150, Abclonal, China, A11109); CD14 (1:200, Proteintech, China, 60253-1-lg); secondary antibodies (1:3000, Proteintech, China, SA00001-1 and SA00001-2).

Techniques: Biomarker Discovery, Expressing

Journal: Journal of Cancer

Article Title: MMP9 and IGFBP1 Regulate Tumor Immune and Drive Tumor Progression in Clear Cell Renal Cell Carcinoma

doi: 10.7150/jca.48664

Figure Lengend Snippet: MMP9 and IGFBP1 were up-regulated in the monocytes low group and negative correlated to CD14. (A-C) Both protein expression and mRNA levels of MMP9/IGFBP1 were elevated in the monocytes low group. (D) The protein expression levels of MMP9 (Pearson r=-0.0.43, P=0.035) and IGFBP1 (Pearson r=-0.39, P=0.047) were negative correlated to CD14.

Article Snippet: The primary antibodies and secondary antibodies were as following: MMP9 (1:100, Abclonal, China, A0289); IGFBP1 (1:150, Abclonal, China, A11109); CD14 (1:200, Proteintech, China, 60253-1-lg); secondary antibodies (1:3000, Proteintech, China, SA00001-1 and SA00001-2).

Techniques: Expressing

Journal: Scientific Reports

Article Title: TLR2-induced surface mobilization and release of CD14 in human platelets

doi: 10.1038/s41598-025-22715-7

Figure Lengend Snippet: Stimulation with a TLR2 agonist induces CD14 expression in WP. WP (3 × 10 8 platelets/mL) were incubated with buffer as control ( a ), with 15 µg/mL LPS ( b ) or with 15 µg/mL Pam3CSK4 ( c ), followed by fluorescent staining with an anti-CD14 antibody or with an isotype IgG. CD14 expression measured by flow cytometry is given as median fluorescence intensity (MFI) in arbitrary units. Representative histograms of flow cytometry are shown on the left side, illustrating the absence ( a, b ) or the shift of fluorescence to the right ( c ). In histograms on the right side, results for CD14 expression are presented as mean of median fluorescence intensity values ± SEM; n = 5; * p < 0.05.

Article Snippet: The polyclonal goat anti-CD14 antibody (Novus Cat# NB100-2807, RRID: AB_10002272) and the isotype control goat IgG (Novus Cat# NB410-28088, RRID: AB_1853319) were from Bio-Techne (Minneapolis, USA).

Techniques: Expressing, Incubation, Control, Staining, Flow Cytometry, Fluorescence

Journal: Scientific Reports

Article Title: TLR2-induced surface mobilization and release of CD14 in human platelets

doi: 10.1038/s41598-025-22715-7

Figure Lengend Snippet: Membrane-bound long isoform of CD14 decreases in TLR2-stimulated WP. Using Western Blot analysis, the amount of membrane-bound CD14 isoforms, long ( a ) and short ( c ), were detected in lysed pellets of human WP (3 × 10 8 platelets/mL) after incubation with buffer (basal), with 15 µg/mL LPS or with 15 µg/mL Pam3CSK4 for 30 min. WM served as positive controls. A representative Western blot figure is shown ( b ). Ponceau S staining was used as loading control and for normalization. Results are given as mean ± SEM in arbitrary units relative to the loading controls; n = 7; * p < 0.05. Original uncropped blots are shown in supplemental figure .

Article Snippet: The polyclonal goat anti-CD14 antibody (Novus Cat# NB100-2807, RRID: AB_10002272) and the isotype control goat IgG (Novus Cat# NB410-28088, RRID: AB_1853319) were from Bio-Techne (Minneapolis, USA).

Techniques: Membrane, Western Blot, Incubation, Staining, Control

Journal: Scientific Reports

Article Title: TLR2-induced surface mobilization and release of CD14 in human platelets

doi: 10.1038/s41598-025-22715-7

Figure Lengend Snippet: CD14 is located inside of unstimulated, not adherent platelets. The figures show ultrathin sections of human WP using transmission electron microscopy. WP were fixed floating in the suspension and stained with an anti-CD14 (12 nm gold) antibody ( a ) or with isotype control ( b ). A highly specific intracellular staining of CD14 ligand is visible inside of WP along the plasma membrane ( a ), whereas no staining is visible in the negative control ( b ); n = 2.

Article Snippet: The polyclonal goat anti-CD14 antibody (Novus Cat# NB100-2807, RRID: AB_10002272) and the isotype control goat IgG (Novus Cat# NB410-28088, RRID: AB_1853319) were from Bio-Techne (Minneapolis, USA).

Techniques: Transmission Assay, Electron Microscopy, Suspension, Staining, Control, Clinical Proteomics, Membrane, Negative Control

Journal: Scientific Reports

Article Title: TLR2-induced surface mobilization and release of CD14 in human platelets

doi: 10.1038/s41598-025-22715-7

Figure Lengend Snippet: CD14 is expressed on the surface and inside of collagen-adherent platelets. WP (2.5 × 10 7 platelets/mL), diluted with HEPES and supplemented with 1 mM CaCl2, were seeded onto collagen-coated slides and left to adhere for 30 min at room temperature. After fixation, without ( a,c ) or with permeabilization ( b,d ), platelets were stained overnight with an Alexa Fluor 488-conjugated anti-CD14 antibody and with a Vioblue-conjugated anti-CD41a antibody as indicated, followed by mounting and subsequent analysis on an inverted Nikon Eclipse Ti2 microscope using a 100 x oil immersion objective and a 1.5x zoom (14-bit digitalization). The images show representative slides with adherent unstimulated platelets; n = 3.

Article Snippet: The polyclonal goat anti-CD14 antibody (Novus Cat# NB100-2807, RRID: AB_10002272) and the isotype control goat IgG (Novus Cat# NB410-28088, RRID: AB_1853319) were from Bio-Techne (Minneapolis, USA).

Techniques: Staining, Microscopy

Journal: Scientific Reports

Article Title: TLR2-induced surface mobilization and release of CD14 in human platelets

doi: 10.1038/s41598-025-22715-7

Figure Lengend Snippet: Stimulation with convulxin induces CD14 expression in WP. WP (3 × 10 8 platelets/mL) were incubated for indicated times with buffer as control or with 100 ng/mL convulxin followed by fluorescent staining with an anti-CD14 antibody or with an isotype IgG antibody, and with Alexa Fluor 488-conjugated donkey anti-goat secondary antibody. Results for CD14 expression measured by flow cytometry are presented as mean of median intensity fluorescence values (MFI) ± SEM; n = 6; * p < 0.05 (compared to the corresponding control).

Article Snippet: The polyclonal goat anti-CD14 antibody (Novus Cat# NB100-2807, RRID: AB_10002272) and the isotype control goat IgG (Novus Cat# NB410-28088, RRID: AB_1853319) were from Bio-Techne (Minneapolis, USA).

Techniques: Expressing, Incubation, Control, Staining, Flow Cytometry, Fluorescence

Journal: Scientific Reports

Article Title: TLR2-induced surface mobilization and release of CD14 in human platelets

doi: 10.1038/s41598-025-22715-7

Figure Lengend Snippet: Co-localization of CD14 with TLR2 and TLR4 in adherent platelets. WP (2.5 × 10 7 platelets/mL), diluted with HEPES and supplemented with 1 mM CaCl 2 , were seeded onto collagen-coated slides and left to adhere for 30 min at room temperature. After fixation, without or with permeabilization, platelets were stained overnight with a goat anti-CD14 antibody and a mouse anti-TLR2 ( A ) or a mouse anti-TLR4 ( B ) antibody as indicated, followed by staining with an Alexa Fluor 488-conjugated donkey anti-goat antibody and a DyLight 550-conjugated donkey anti-mouse antibody, mounting and subsequent analysis on an inverted Nikon Eclipse Ti2 microscope using a 100 x oil immersion objective and a 1.5 x zoom (14-bit digitalization). The images show representative slides with adherent unstimulated platelets; n = 5.

Article Snippet: The polyclonal goat anti-CD14 antibody (Novus Cat# NB100-2807, RRID: AB_10002272) and the isotype control goat IgG (Novus Cat# NB410-28088, RRID: AB_1853319) were from Bio-Techne (Minneapolis, USA).

Techniques: Staining, Microscopy

Journal: Scientific Reports

Article Title: TLR2-induced surface mobilization and release of CD14 in human platelets

doi: 10.1038/s41598-025-22715-7

Figure Lengend Snippet: Mechanism of CD14 regulation in human platelets. Collagen-adherent platelets express CD14 on their surface and TLR2-stimulated platelets release sCD14. The figure was created with the software BioRender (URL: https://biorender.com , version used on 2025-05-30; Created in BioRender. Kobsar, A. (2025) https://BioRender.com/ammeo3s ).

Article Snippet: The polyclonal goat anti-CD14 antibody (Novus Cat# NB100-2807, RRID: AB_10002272) and the isotype control goat IgG (Novus Cat# NB410-28088, RRID: AB_1853319) were from Bio-Techne (Minneapolis, USA).

Techniques: Software