terminal complement complex hk 106 assay  (Hycult Biotech)

Journal: Acta Pharmaceutica Sinica. B

Article Title: Ginsenoside modified lipid-coated perfluorocarbon nanodroplets: A novel approach to reduce complement protein adsorption and prolong in vivo circulation

doi: 10.1016/j.apsb.2023.11.016

Figure Lengend Snippet: Influence of complement-mediated surface protein corona (PC) on phagocytosis of lp-NDs: formation and characterization. (A) Heat map of the top 30 plasma proteins identified as components of the PC formed on lp-NDs. The values in heat map indicate the logarithmically transformed label-free quantification (LFQ) detected by LC–MS, multiplied by 2. #1–3 indicate three distinct plasma samples. (B) Top 20 KEGG enrichment pathways. (C) Inhibition of complement pathway by different blockers: EDTA inhibits all pathways, EGTA/Mg 2+ specifically inhibits Ca 2+ -sensitive pathways, anti-C1q antibody inhibits the classical pathway (CP), anti-properdin (anti-P) antibody inhibits the alternative pathway (AP), and PBS was used as the control group. C3 levels in the PC are quantified by enzyme-linked immunosorbent assay (ELISA). The accompanying graph illustrates the standard curve for C3. (D) Following the inhibition of complement pathway by different blockers, the SC5b-9 levels in plasma incubated with lp-NDs are assessed by ELISA. The accompanying graph displays the standard curve for SC5b-9. (E) Effects of complement components on phagocytosis observed via confocal laser scanning microscopy. Three groups: bare lp-NDs, lp-NDs@PC [C−] (lp-NDs with protein corona lacking complement components) and lp-NDs@PC [C+] (lp-NDs with a protein corona containing complete complement components) interact with macrophages for 10, 30, 60 and 120 min, respectively. The lp-NDs are labeled with DiI (red), nucleus are labeled with Hoechst (blue) and lysosomes are labeled with Lyso-tracker (green). Single cell (arrow) higher magnification boxed areas are shown on the upper right, beneath color scatter plots corresponding to the whole images, line-scan intensity curves corresponding to the single cell images (arrow). The intracellular co-localization between lp-NDs and lysosomes is assessed using the Pearson correlation coefficient (PCC). Scale bar = 10 μm. (F) Quantification of Thp-1 cells' uptake based on fluorescence intensity of per cell (a.u.) obtained from confocal laser scanning microscopy images of bare lp-NDs, lp-NDs@PC [C-] and lp-NDs@PC [C+] at 10, 30, 60, and 120 min. (G) Flow cytometry mean fluorescence intensity (MFI) of cellular uptake of bare lp-NDs, lp-NDs@PC [C-] and lp-NDs@PC [C+] at 10, 30, 60, and 120 min. Data are presented as mean ± SD ( n = 3), ns, not significant. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ∗∗∗∗ P < 0.0001.

Article Snippet: The SC5b-9 levels in human plasma and rat plasma were determined using ELISA kits specific for humans (MicroVue SC5b-9 Plus EIA, Quidel Corporation, San Diego, CA, USA) and rats (Hycult Biotech, HK106-01, Uden, Netherlands), respectively, following the manufacturer's instructions.

Techniques: Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Inhibition, Control, Enzyme-linked Immunosorbent Assay, Incubation, Confocal Laser Scanning Microscopy, Labeling, Fluorescence, Flow Cytometry

Journal: Acta Pharmaceutica Sinica. B

Article Title: Ginsenoside modified lipid-coated perfluorocarbon nanodroplets: A novel approach to reduce complement protein adsorption and prolong in vivo circulation

doi: 10.1016/j.apsb.2023.11.016

Figure Lengend Snippet: Anti-complement and anti-macrophage effect of Gs lp-NDs. (A) Top 30 plasma proteins identified by PC LC–MS analysis of C lp-NDs, 25% Rd lp-NDs, and 25% Rh2 lp-NDs. The values in heat map indicates the logarithmically transformed label-free quantification (LFQ) multiplied by 2. #1–3 indicates three different plasma samples. (B) The LFQ of C3 in C lp-NDs, 25% Rd lp-NDs, and 25% Rh2 lp-NDs. (C) In vitro human C3 in PC of C lp-NDs, 25% Rd lp-NDs and 25% Rh2 lp-NDs. (D) In vitro human SC5b-9 generation following plasma incubation with C lp-NDs, 25% Rd lp-NDs, and 25% Rh2 lp-NDs. (E) In vivo SC5b-9 production in rats following injection with C lp-NDs, 25% Rd lp-NDs, and 25% Rh2 lp-NDs. (F) Uptake of C lp-NDs and Gs lp-NDs by Thp-1 cells. Lysosomes of THP-1 cells, cell nuclei, and lp-NDs are labeled with green, blue, and red fluorescent dye, respectively. Scale bar = 50 μm. (G) Quantification of Thp-1 cell uptake based on mean fluorescence intensity (MFI) values of each cell obtained from confocal laser scanning microscopy (CLSM) images. (H) Cell counts/fluorescence intensity histogram and relevant MFI of Thp-1 cells uptake C lp-NDs and Gs lp-NDs, determined via flow cytometry. (I) Internalization of C lp-NDs and Gs lp-NDs by peripheral blood macrophages (PBMs). Lysosomes within PBMs cells, cell nuclei, and lp-NDs are fluorescently labeled with green, blue, and red dyes, respectively. Scale bar = 20 μm. (J) Quantification of PBMs cell uptake by determining the MFI values for each cell, based on analysis of CLSM images. (K) Flow cytometer analysis of the histogram depicting PBMs cell count and fluorescence intensity, along with the corresponding MFI, illustrating the uptake of C lp-NDs and Gs lp-NDs by PBMs. Data are presented as mean ± SD ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ∗∗∗∗ P < 0.0001.

Article Snippet: The SC5b-9 levels in human plasma and rat plasma were determined using ELISA kits specific for humans (MicroVue SC5b-9 Plus EIA, Quidel Corporation, San Diego, CA, USA) and rats (Hycult Biotech, HK106-01, Uden, Netherlands), respectively, following the manufacturer's instructions.

Techniques: Liquid Chromatography with Mass Spectroscopy, Transformation Assay, In Vitro, Incubation, In Vivo, Injection, Labeling, Fluorescence, Confocal Laser Scanning Microscopy, Flow Cytometry, Cell Counting