quality control beads  (NanoFCM Inc)

Journal: Journal of Extracellular Vesicles

Article Title: Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs)

doi: 10.1002/jev2.12520

Figure Lengend Snippet: Quantitative analysis of intensity statistics in the three fluorescent labels on individual sEVPs from different subpopulaitons. (a) Intensity scatter plots between each two of the three colocalized fluorescent markers in CFSE + CD63 + sEVs (include 1432 particles from two representative trials). (b) Intensity scatter plots between two colocalized fluorescent markers in CFSE + CD63 + NPs, CFSE − CD63 + sEVs, and CFSE + CD63 − sEVs, respectively (include 2314, 298, and 1150 particles from two representative trials, respectively). (c) Intensity histograms of single PE‐Cy5 labelled CD63 antibodies and PE‐Cy5 in individual CFSE + CD63 + sEVs are used to calculate histograms of CD63 count per CFSE + CD63 + sEVs. Particle intensity profiles from all trials are included here.

Article Snippet: The instrument underwent calibration per the manufacturer's protocols, employing 250 nm fluorescent quality control beads of known concentration and S16M‐Exo sizing beads comprising four distinct‐sized silica nanospheres (NanoFCM Inc.) with diameters of 68, 91, 113, and 155 nm.

Techniques:

Journal: Journal of Extracellular Vesicles

Article Title: Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs)

doi: 10.1002/jev2.12520

Figure Lengend Snippet: Average signal of the three fluorescent labels per sEVPs in various particle subpopulations derived from four cell types (a) Average CD63 number per particle in four CD63‐positive sEVP subgroups. (b) Average integrated CFSE fluorescence intensity per particle in four CFSE‐positive sEVP subgroups. (c) Average integrated COE‐Ben fluorescence intensity per particle in four COE‐Ben‐positive sEVP subgroups. (d) Signal variance of the normalized intensity of each fluorescent label among all sEVPs subpopulations from four cell types. * p < 0.05, ** p < 0.01. At least three individual imaging experiments were performed with three batches of PC3, HEK, A549, and RBC sEVP samples. For each sample, ten to fifteen 100 µm × 100 µm regions were randomly selected, and all particles (31380 particles for PC3, 21200 particles for HEK, 23411 particles for A549, and 20183 particles for RBC) in these regions were sampled and analysed. Error bars represent standard error among different trials.

Article Snippet: The instrument underwent calibration per the manufacturer's protocols, employing 250 nm fluorescent quality control beads of known concentration and S16M‐Exo sizing beads comprising four distinct‐sized silica nanospheres (NanoFCM Inc.) with diameters of 68, 91, 113, and 155 nm.

Techniques: Derivative Assay, Fluorescence, Imaging

Journal: Journal of Extracellular Vesicles

Article Title: Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs)

doi: 10.1002/jev2.12520

Figure Lengend Snippet: Identification and quantification of a robust fluorescent marker for lipid membranes. (a) Schematic demonstration of staining fluorescent SUVs (containing either RhoB‐PE or Cy5‐PE) with water‐soluble membrane probes (CellVue Claret, PKH26, or COE‐Ben) before surface anchoring to a PLL‐PEG‐biotin and neutravidin functionalized substrate for TIRF imaging. (b) Representative TIRF image (grayscale) and 3D intensity plots (two‐channel merged) of SUVs stained with both water‐soluble membrane probes and fluorescent lipid‐dye conjugates. (c) Colocalization analysis of the RhoB intensities and the COE‐Ben intensities per SUV, including quantification of subpopulations and intensity scatter plots of the two fluorescent markers. The composition of SUVs is: 0.025 mol% RhoB‐PE, 0.5 mol% biotin‐PEG2000‐DSPE, 10 mol% DOPG, and 89.475 mol% DOPC, or 0.5 mol% Cy5‐PE, 0.5 mol% biotin‐PEG2000‐DSPE, 10 mol% DOPG, and 89 mol% DOPC. Three individual imaging experiments were performed with three different batches of SUV samples. Seven 100 µm × 100 µm regions were randomly selected, and all 6015 particles in these regions were sampled and analysed.

Article Snippet: The instrument underwent calibration per the manufacturer's protocols, employing 250 nm fluorescent quality control beads of known concentration and S16M‐Exo sizing beads comprising four distinct‐sized silica nanospheres (NanoFCM Inc.) with diameters of 68, 91, 113, and 155 nm.

Techniques: Marker, Staining, Membrane, Imaging

Journal: Journal of Extracellular Vesicles

Article Title: Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs)

doi: 10.1002/jev2.12520

Figure Lengend Snippet: Demonstration of the entire workflow for fluorescent labelling and surface anchoring of sEVPs that enables high‐content TIRF imaging at the single‐particle level. (a) Schematic illustration of the staining order to label sEVPs with three different fluorescent markers—CFSE, PE‐Cy5 labelled CD63 antibodies, and COE‐Ben. (b) Representative TIRF images of PC3 sEVPs in the three fluorescent channels, shown as both individuals and as merged. (c) Density of surface‐anchored sEVPs, respectively detected in the three fluorescence channels, are plotted as a function of NeuA concentration. Two individual imaging experiments were performed with two different batches of PC3 sEVP samples for each NeuA concentration. At each NeuA concentration, eight 100 µm x 100 µm regions were randomly selected, and all particles in these regions were sampled to calculate the average particle density. Error bars represent standard error among selected regions. (d) Surface densities and captured fractions of biotin‐containing SUVs and sEVPs derived from PC3, HEK293, A549, and RBC. sEVPs in all three fluorescence channels are included. Two individual imaging experiments were performed with one batch of SUV, PC3 sEVP, HEK sEVP, A549 sEVP, and RBC sEVP. For each sample, six 100 µm × 100 µm regions were randomly selected, and all particles in these regions were sampled to calculate the average particle density and the corresponding captured fraction. Error bars represent standard error among selected regions.

Article Snippet: The instrument underwent calibration per the manufacturer's protocols, employing 250 nm fluorescent quality control beads of known concentration and S16M‐Exo sizing beads comprising four distinct‐sized silica nanospheres (NanoFCM Inc.) with diameters of 68, 91, 113, and 155 nm.

Techniques: Imaging, Single Particle, Staining, Fluorescence, Concentration Assay, Derivative Assay

Journal: Journal of Extracellular Vesicles

Article Title: Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs)

doi: 10.1002/jev2.12520

Figure Lengend Snippet: Identification and characterization of distinct subpopulations within sEVP samples. (a) Demonstration of the seven distinct subpopulations when sEVPs are labelled with three fluorescence markers in a multidimensional manner. The 3D intensity surface maps in the three fluorescent marker channels plotted under the same column belong to one representative particle of a specific subpopulation. (b) Venn diagrams of subpopulation fractions in four different sEVP samples. At least three individual imaging experiments were performed with three batches of PC3, HEK, A549, and RBC sEVP samples. For each sample, ten to fifteen 100 µm × 100 µm regions were randomly selected, and all particles (31380 particles for PC3, 21200 particles for HEK, 23411 particles for A549, and 20183 particles for RBC) in these regions were sampled and analysed.

Article Snippet: The instrument underwent calibration per the manufacturer's protocols, employing 250 nm fluorescent quality control beads of known concentration and S16M‐Exo sizing beads comprising four distinct‐sized silica nanospheres (NanoFCM Inc.) with diameters of 68, 91, 113, and 155 nm.

Techniques: Fluorescence, Marker, Imaging

Journal: STAR Protocols

Article Title: Protocol for separation of fungal extracellular vesicles using ultracentrifugation from solid medium cultures

doi: 10.1016/j.xpro.2024.103069

Figure Lengend Snippet:

Article Snippet: NanoFCM quality control nanospheres (QC beads), 250 ± 5 nm, 2E+10 particles/mL , NanoFCM Co. Ltd. , QS2503.

Techniques: Recombinant, Amplex Red Cholesterol Assay, Software, Centrifugation, Suction Filtration, Membrane