Journal: Cardiovascular Diabetology

Article Title: Adiponectin levels and expression of adiponectin receptors in isolated monocytes from overweight patients with coronary artery disease

doi: 10.1186/1475-2840-10-14

Figure Lengend Snippet: Flow cytometric analysis of adiponectin receptors in isolated monocytes . (A) Dot plot presentation of isolated mononuclear cells. Cells subpopulations were distinguished by granularity (side scatter) versus staining with CD14-PE monoclonal antibody. Monocytes (CD14-PE + ) were gated (R). (B) Analysis of Adipo-R2 log fluorescence of the gated monocytes. (C) Dot plot of the Adipo-R2 expression versus CD14-PE. The upper right quadrant includes the cells showing positive expression for both adipoR2 and CD14 antibodies (green colour).

Article Snippet: The monocyte fraction was simultaneously stained with anti-CD14-PE monoclonal antibody (BD Biosciences, San Jose, CA, USA).

Techniques: Isolation, Staining, Fluorescence, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Flotillins affect LPS-induced TLR4 signaling by modulating the trafficking and abundance of CD14

doi: 10.1007/s00018-024-05221-3

Figure Lengend Snippet: The effect of Flot2 knock-down on cell-surface CD14 abundance, endocytosis and shedding. Cells were transfected with Flot2 -specific shRNA variant No. 5 (Flot2 shRNA) or control shRNA NC1 (NC shRNA). A , B The cell-surface level of CD14 determined by flow cytometry ( A ) and expressed as percentage of the value in control cells ( B ). C Fractionation of Triton X-100 lysates of the cells. TX-100 sol.—fraction soluble in 0.1% Triton X-100, TX-100 insol.—fraction insoluble in Triton X-100, SDS—fraction soluble in 4% SDS. Numbers below blots show the relative content of the respective protein in each fraction determined by densitometry (OD). Results of one representative experiment of two run in duplicate are shown. D Scheme of the biotinylation-based assay of CD14 endocytosis and shedding. E Constitutive endocytosis of CD14 and TfR determined by immunoblotting (left panel) followed by densitometric analysis relative to actin content in the input samples (right panel). Three different amounts of the initial cell surface fraction of proteins were subjected to SDS-PAGE and after averaging, they were used as a reference point to calculate the percentage of CD14 and TfR internalized after 30–90 min and also the amount of sCD14 shed from the cell surface ( F ). In E , to balance the signal from CD14 between control and flotillin-depleted cells, different portions of cellular fractions (of the original 70 μl) were subjected to SDS-PAGE, as indicated above blots. Positions of molecular weight markers are shown on the right in kDa. The total amount of CD14 and TfR relative to actin in input cell lysates is shown in Supplementary Fig. S2. F , G The content of sCD14 in culture supernatants determined by the biotinylation assay ( F ) and by ELISA prior to and after stimulation of cells with 10 ng/ml LPS for 1 or 4 h at 37 °C ( G ). Data shown in B , E , F , G are mean ± SD from three or four experiments. Significantly different values as indicated by Student's t -test ( B , E , F ) and 1-way ANOVA with Tukey’s post hoc test ( G ) are marked

Article Snippet: Total amount of sCD14 was determined in cell supernatants using a mouse ELISA kit (R&D System) according to the manufacturer’s instructions.

Techniques: Knockdown, Transfection, shRNA, Variant Assay, Control, Flow Cytometry, Fractionation, Cell Surface Biotinylation Assay, Western Blot, SDS Page, Molecular Weight, Enzyme-linked Immunosorbent Assay