Journal: Molecular Neurobiology

Article Title: Epigallocatechin Gallate Modulates Microglia Phenotype to Suppress Pro-inflammatory Signalling Cues and Inhibit Phagocytosis

doi: 10.1007/s12035-023-03845-3

Figure Lengend Snippet: EGCG blocks microglia phagocytosis of Zymosan particles. A Representative immunofluorescence images of primary microglia live-stained with a nuclear marker (DAPI; blue) and a cell membrane marker (CellMask; magenta), showing phagocytosis of pHrodo Green Zymosan bioparticles (green) at different timepoints. Individual rows represent individual EGCG concentrations. White boxes indicate inset images on the right panel. Scale bar = 50 µm. B Quantified time course data of average cellular 488-fluorescence intensity, grouped by EGCG concentration and normalised relative to Vehicle (average of 1.5 h and 2 h timepoints). Datapoints and error bars indicate mean ± S.E.M. n = 4. C Quantified time course data of proportion of cells exhibiting phagocytosis, grouped by EGCG concentration and normalised relative to Vehicle (average of 1.5-h and 2-h timepoints). Datapoints and error bars indicate mean ± S.E.M. n = 4. D Quantified time course data of average cellular 488-fluorescence intensity for 2,2′-dipyridyl treated microglia internalising HiLyte Fluor488 labelled Aβ42 peptide, normalised relative to Vehicle (average of 1.5-h and 2-h timepoints). Datapoints and error bars indicate mean ± S.E.M n = 4. C Quantified time course data of average cellular 488-fluorescence intensity for 2,2′-dipyridyl treated microglia internalising pHrodo Green Zymosan bioparticles, normalised relative to Vehicle (average of 1.5-h and 2-h timepoints). Datapoints and error bars indicate mean ± S.E.M n = 4

Article Snippet: D-PBS was added to prepare a 100 µM working solution that was then left to incubate at room temperature for 2 h. pHrodo Green Zymosan Bioparticles (#P35365, Fisher Scientific) were resuspended to 0.5 mg/ml in Live Cell Imaging Solution (#12363603, Fisher Scientific), followed by alternate trituration and vortexing for 10 min, immediately prior to use.

Techniques: Immunofluorescence, Staining, Marker, Membrane, Fluorescence, Concentration Assay

Journal: Nature

Article Title: Regulation of the mammalian-brain V-ATPase through ultraslow mode-switching

doi: 10.1038/s41586-022-05472-9

Figure Lengend Snippet: a, An illustration of the assay for single-transporter activity recordings. Proteoliposomes, carrying the pH-sensitive fluorophore pHrodo, syntaxin 1 and SNAP25, are immobilized on a glass slide. SVs containing mostly one copy of the V-ATPase are added in solution and fuse with the proteoliposomes, producing SVh. On ATP hydrolysis, the V-ATPase pumps protons into the lumen of SVh. b, Ensemble average of acidification kinetics from about 340 single SVh (red) and about 104inactive vesicles (grey). The data are not corrected for photobleaching. Proton-pumping and vesicle acidification were initiated by addition of ATP and inhibited by 200 nM bafilomycin. The solid lines are the mean; the shaded area corresponds to one standard deviation (s.d.). About 4% of vesicles showed ATP-dependent acidification. c, Left, an ultralong-term acidification trace of a representative single SVh revealing that the V-ATPase stochastically switches between long-lived proton-pumping and inactive modes (red trace). The black trace corresponds to a representative inactive vesicle. Right, histograms representing the corresponding distribution of intensities. PDF, probability density function. d, The top two traces show SVh with one acidification plateau corresponding to one active V-ATPase per SVh. The bottom two traces are representative of SVh with respectively two and three acidification plateaus, corresponding to two and three active V-ATPases. The kinetics have been processed by a Chung–Kennedy filter54 for visual clarity, unless stated otherwise. e, Bar chart of the number of active V-ATPases per SVh based on step-counting acidification plateaus; the average number is 1.2 ± 0.1. The error bars correspond to one s.d. n = 8; hereafter, n is the number of independent experiments.

Article Snippet: Subsequently, the acidification of the pHrodo-labelled hybrid SVs was monitored by measuring changes in fluorescence at 590 nm (slits: 3 nm for excitation at 560 nm and 5 nm for emission at 590 nm) in a fluorimeter (Fluoromax-2, Horiba) at 37 °C.

Techniques: Activity Assay, Standard Deviation

Journal: Vaccines

Article Title: Patients with SARS-CoV-2-Induced Viral Sepsis Simultaneously Show Immune Activation, Impaired Immune Function and a Procoagulatory Disease State

doi: 10.3390/vaccines11020435

Figure Lengend Snippet: COVID-19 patients show impaired phagocytic activity of monocytes and granulocytes. Whole blood of healthy controls, non-severe COVID-19 and severe COVID-19 patients was incubated with E.coli -pHrodo (10μL of a 1 mg/mL solution), which is fluorescent only at low pH, i.e., after active phagocytosis. After 30 min incubation cells were analyzed for ( a ) + ( b ) phagocytosis and ( c ) + ( d ) upregulation of CD11b on ( a ) + ( c ) monocytes and ( b ) + ( d ) granulocytes. Depicted are differences (delta) of mean fluorescence intensities (MFI) between unstimulated controls and cells stimulated with E.coli -pHrodo. Statistical comparison between groups was done by the Kruskal–Wallis test. Unless marked with a p -value, all changes observed are not significant; * p < 0.05; ** p < 0.01; *** p < 0.0001.

Article Snippet: A 50 μL volume of blood was incubated for 1 h with 10 μL of E. coli -pHrodo (Fisher Scientific, 1 mg/mL).

Techniques: Activity Assay, Incubation, Fluorescence, Comparison