macsplex buffer  (Miltenyi Biotec)

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicles are the primary source of blood‐borne tumour‐derived mutant KRAS DNA early in pancreatic cancer

doi: 10.1002/jev2.12142

Figure Lengend Snippet: Differential centrifugation of whole blood efficiently separates different cell and vesicle populations. (a) Schematic of the differential centrifugation protocol used to separate red (RBC) and white blood cells (WBC), platelets (PL), apoptotic bodies (AB), large (LEV) and small extracellular vesicles (SEV), as well as soluble protein (SP) and flow through (FT). (b) Representative transmission electron microscopy images of vesicles enriched in AB, LEV and SEV fractions. Corresponding scale bars are inset. (c) Representative Western blots of blood components for histone H3 (H3), CD42A, cleaved Caspase 9 (CASP9), BAX, CD9 and CD81. A CD9 blotted membrane with vesicular and SP components was additionally overexposed. Molecular weight is indicated beside each blot. (d) Nanoparticle tracking analysis data showing the concentration of particles/ml at sizes ranging from 0–1 μm in LEV, SEV and SP fractions. Inset is a quantification of the concentration of particles from 400–600 nm found in each fraction. (d) Median CD9/63/81 fluorescence for 35 capture antibody coated beads from the MACSplex vesicle surface protein flow cytometry assay in the different blood components. The platelet fraction was diluted 1:10 before analysis

Article Snippet: Beads were washed by adding 200 μl of MACSPlex Buffer (MBP) to each well and the filter plate was put on a vacuum manifold with vacuum applied at ‐100 mBar (Sigma‐Aldrich, Germany) until all wells were drained.

Techniques: Centrifugation, Transmission Assay, Electron Microscopy, Western Blot, Molecular Weight, Concentration Assay, Fluorescence, Flow Cytometry

Journal: Biomedicines

Article Title: Profiling Inflammatory Extracellular Vesicles in Plasma and Cerebrospinal Fluid: An Optimized Diagnostic Model for Parkinson's Disease.

doi: 10.3390/biomedicines9030230

Figure Lengend Snippet: Figure 1. Plasma and CSF preparation for MACSPlex human Exosome assay. (a) Protocol for extracellular vesicle (EV) enrichment and characterization by MACSPlex Exosome Assay. Blood and cerebrospinal fluid (CSF) underwent serial centrifugation to eliminate cellular components and larger EVs. Samples were incubated overnight with phycoerythrin (PE)- and fluorescein isothiocyanate (FITC)-labeled capture beads, coated with antibodies against 37 different EV surface markers. APC-conjugated detection antibodies against CD9, CD63, and CD81 were added and incubated for 1 h. After washing steps, samples were analyzed by flow cytometry. (b) Schematic representation of the 37 EV surface markers analyzed by MACSPlex human Exosome assay.

Article Snippet: In total, 60 μL of plasma and 30 μL of ultracentrifuged CSF were added to the MACSPlex Buffer solution (final volume 120 μL) and analyzed with MACSQuant Analyzer-10 flow cytometer (Miltenyi, Bergisch Gladbach, Germany).

Techniques: Clinical Proteomics, Centrifugation, Incubation, Labeling, Cytometry

Journal: Biomedicines

Article Title: Profiling Inflammatory Extracellular Vesicles in Plasma and Cerebrospinal Fluid: An Optimized Diagnostic Model for Parkinson's Disease.

doi: 10.3390/biomedicines9030230

Figure Lengend Snippet: Figure 3. CSF-derived EVs characterization. Characterization of cerebrospinal fluid (CSF)-derived extracellular vesicles (EVs) by nanoparticle tracking analysis (NTA) and MACSPlex human exosome assay flow cytometry. Healthy controls (HC) were compared with patients with Parkinson’s disease (PD) multisystem atrophy (MSA), or atypical parkinsonism with tauopathies (AP-Tau). (a) EV concentration (n/mL CSF) at NTA. (b) EV diameter (nm) at NTA. Boxplots show median and interquartile range; bars show minimum and maximum values (* p < 0.05). (c) Normalized median fluorescence intensity (nMFI; %) for 37 EV surface markers. Data and statistics are reported in Table S1. (d,e) Different perspectives of 3D-canonical plot reporting patient discrimination according to EV surface marker expression (each patient is indicated by a point and diagnoses are represented by colors: HC, blue; PD, red; MSA, orange; AP-Tau, grey). Canonical axes of the plot (canonical components 1, 2, and 3) are defined by linear discrimination analysis from weighted linear combinations of the 37 EV markers analyzed by flow cytometry. Spheres include patients with linear combination coefficients that fall within the mean ± SD (canonicals 1, 2, and 3 ± SD).

Article Snippet: In total, 60 μL of plasma and 30 μL of ultracentrifuged CSF were added to the MACSPlex Buffer solution (final volume 120 μL) and analyzed with MACSQuant Analyzer-10 flow cytometer (Miltenyi, Bergisch Gladbach, Germany).

Techniques: Derivative Assay, Cytometry, Concentration Assay, Marker, Expressing

Journal: Biomedicines

Article Title: Profiling Inflammatory Extracellular Vesicles in Plasma and Cerebrospinal Fluid: An Optimized Diagnostic Model for Parkinson's Disease.

doi: 10.3390/biomedicines9030230

Figure Lengend Snippet: Figure 4. EV quantitative analysis: plasma vs. CSF. Quantitative analysis of extracellular vesicles (EVs) by nanoparticle tracking analysis (NTA) and MACSPlex assay flow cytometry; plasma samples were compared to paired cerebrospinal fluid (CSF) samples in patients with PD (Parkinson’s disease; n = 4), MSA (multisystem atrophy; n = 4) and AP-Tau (atypical parkinsonism with tauopathies; n = 4). (a) EV concentration (n/mL plasma or CSF) at NTA. (b) EV diameter (nm) at NTA. (c) MFI (expressed as arbitrary unity; a.u.) for CD9, CD63, CD81 and CD9-CD63-CD81 at flow cytometry. Boxplots show median and interquartile range; bars show minimum and maximum values (** p < 0.05; *** p < 0.001). Data and statistics are reported in Table S2.

Article Snippet: In total, 60 μL of plasma and 30 μL of ultracentrifuged CSF were added to the MACSPlex Buffer solution (final volume 120 μL) and analyzed with MACSQuant Analyzer-10 flow cytometer (Miltenyi, Bergisch Gladbach, Germany).

Techniques: Clinical Proteomics, Cytometry, Concentration Assay