Mouse whole blood was treated with either 10 μM ADP or 1.0x105 pg p24 EcoHIV for 30 min, 1 hr, and 2 hrs at 37°C. Subsequently, blood was processed and analyzed for platelet activation by flow cytometry. Briefly, whole blood was fixed with 4% paraformaldehyde and washed twice with 1 ml staining buffer (1x PBS containing 2% BSA). Red blood cells (RBCs) were then lysed using ACK lysis buffer (Life Technologies, Grand Island, NY) and the remaining cells were washed twice in staining buffer. Cells were stained with 2.5 μl anti-mouse CD61-PE (AbD Serotec, Oxford, U.K.) and 1 μl anti-mouse CD62P-FITC (BD Biosciences, San Jose, CA). Following the staining, samples were acquired using a flow cytometer (Accuri C6; Accuri Cytometers, Ann Arbor, MI). Platelets were gated based on forward and side scatter, followed by assessment of the platelet activation marker CD62P. Sizing beads (Mega Mix; BioCytex, Marseille, France) were used to delineate the platelet gate (0.9–3 μm). Unstained cells and cells stained with only CD61 were used as controls. 20,000 events were acquired to measure platelet percentages and activation in terms of CD62P expression.
Megamix
Manufactured by Biocytex
Sourced in France
Megamix is a laboratory mixer designed for efficient and consistent sample homogenization. It features variable speed control and a robust construction for reliable performance in various laboratory settings.
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Lab products found in correlation
Fix perm buffer, by BioLegend (2 mentions)
Anti mouse cd62p fitc, by BD (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
α6 integrin, by BioLegend (1 mentions)
Canto 2, by BD (1 mentions)
Glycophorin a pe, by BD (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Flow count, by Beckman Coulter (1 mentions)
Guava easycyte flow cytometer, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Navios flow cytometer, by Beckman Coulter (1 mentions)
Annexin 5 binding buffer, by BioLegend (1 mentions)
Annexin 5, by BioLegend (1 mentions)
Facscan flow cytometer, by Beckman Coulter (1 mentions)
Perfect count microspheres, by Cytognos (1 mentions)
Sodium citrate, by BD (1 mentions)
Trucount beads, by BD (1 mentions)
Isoflow sheath fluid, by Beckman Coulter (1 mentions)
Cxp analysis, by Beckman Coulter (1 mentions)
Cxp acquisition, by Beckman Coulter (1 mentions)
11 protocols using megamix
1
Platelet Activation by ADP and EcoHIV
2
Multiparametric Flow Cytometry Analysis
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MPs were examined by flow cytometry analyses (BD Canto II, USA). The instrument was rinsed with particle-free rinse solution for 15 minutes to eliminate the background. Different sized beads (Mega-mix, 0.5, 0.9, 3 µm, Biocytex, Marsille, France) represented size markers, and a log scale for forward scatter and side scatter parameters were used for analysis. FACS analysis was performed as described previously [14] (link), [18] (link), [19] (link) using the following FITC- or PE-conjugated antibodies: CD29, CD44 (BD science), CD73, α4 integrin, α5 integrin, and α6 integrin (Biolegend, San Diego, CA). FITC- or PE- mouse non-immune isotypic IgG were used as a control.
3
Fluorescent Bead-Based Assay Protocol
4
Multicolor Flow Cytometry for MP Enumeration
5
Isolation and Characterization of MSC-Derived Extracellular Vesicles
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EV were obtained from supernatants of MSC at 80% confluence, as previously described 26 . Briefly, MSC were cultured overnight in D‐MEM (Gibco, Life Technologies, Milan, Italy) without foetal calf serum (FCS). Supernatants were centrifuged at 3,000 × g for 20 min. to remove cellular debris, and cell‐free supernatants were then centrifuged twice at 100,000 × g for 1 hr at 4°C.
Fluorescent beads ranging in size from 0.1 to 1 μm (Megamix; BioCytex, Marseille, France) were employed to precisely gate EV. As EV derived from MSC express surface molecules that are characteristic of the cells of origin, anti‐rat CD49e (as positive marker) and anti‐rat CD45 (as negative marker) (both from BioLegend) were used. The analysis was performed by direct immunofluorescence with a Navios flow cytometer (Beckman Coulter), and the data were analysed using Kaluza software. Moreover, some specific exosomal markers, such as CD63, CD9 and CD81 (Miltenyi Biotec, Bergisch Gladbach, Germany), were also analysed, using the Guava easyCyte FlowCytometer (Millipore, Billerica, MA, USA) with InCyte software.
Fluorescent beads ranging in size from 0.1 to 1 μm (Megamix; BioCytex, Marseille, France) were employed to precisely gate EV. As EV derived from MSC express surface molecules that are characteristic of the cells of origin, anti‐rat CD49e (as positive marker) and anti‐rat CD45 (as negative marker) (both from BioLegend) were used. The analysis was performed by direct immunofluorescence with a Navios flow cytometer (Beckman Coulter), and the data were analysed using Kaluza software. Moreover, some specific exosomal markers, such as CD63, CD9 and CD81 (Miltenyi Biotec, Bergisch Gladbach, Germany), were also analysed, using the Guava easyCyte FlowCytometer (Millipore, Billerica, MA, USA) with InCyte software.
6
Quantification of Platelet-Derived Microparticles
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Blood sample was collected into a 5 mL tube containing 3.2% sodium citrate (BD, Plymouth, UK) and preparation-measurement of PMPs was performed at room temperature within 4 h post-collection. PMPs were distinguished from non-platelets events in platelet poor plasma by their expression of the surface marker Glycoprotein IIIa (CD61) using PE anti-human CD61 monoclonal antibody (BioLegend) and their binding capacity with Annexin-V using FITC conjugated, Annexin-V (BioLegend). They were distinguished from platelets by forward scatter to that of fluorescence-labeled reference beads reagent (Megamix, Biocytex) which allowed standardizing the set-up of PMPs analysis region (0.5-1 μm) and guaranteeing the stability of the settings [15 (link)]. The number of CD61 and Annexin-V double-positive events was calculated relative to the number of beads added to samples (Perfect count microspheres, Cytognos) and were expressed as events/μL. To avoid unspecific antibody binding, Annexin-V binding buffer (Biolegend) was also used. Antibody solutions were centrifuged prior to FACS to avoid artifacts due to aggregation. Flow cytometric analysis was performed with a FACScan flow cytometer (Coulter).
7
Flow Cytometric Analysis of Immune Complexes
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Immune complexes were analyzed on the flow cytometer, Cytomics FCM 500 from Beckman Coulter 22, Avenue des Nations CS 54359, 93420, Villepinte. It is equipped with 2 lasers 488 nm, 40 mW and 638 nm, 25 mW. It has the ability to analyze events on five colors: 525 nm, 575 nm, 620 nm, 675/695 nm, and 755 nm.
The sheath fluid for the cytometer was IsoFlow™ Sheath Fluid from Beckman Coulter, Part Number 8448010, an isotonic fluid at a pH 7.35-7.65, with Sodium Phosphate Dibasic, Sodium Fluoride, Diethylene Glycol Phenyl Ether, and 2-Phenoxyethanol.
A microparticle gate was established in agreement with the published method for characterization of circulating cell-derived microparticles by preliminary standardization experiments using a blend of size-calibrated 0.3, 0.5, 0.9, and 3 μm fluorescent beads, Megamix®, Biocytex, Marseille, France [27 (link), 28 (link)]. To enlarge the scale of fluorescent beads, in some experiments, Trucount beads (Beckton Dickinson) of 10 μm were added.
A gate for DNA-anti-DNA immune complexes for each series of experiments was built around DNA-anti-DNA immune complexes observed in the aliquot of the pool of 15 SLE serum samples incubated with calf thymus DNA. See below for more details and for compensations.
The sheath fluid for the cytometer was IsoFlow™ Sheath Fluid from Beckman Coulter, Part Number 8448010, an isotonic fluid at a pH 7.35-7.65, with Sodium Phosphate Dibasic, Sodium Fluoride, Diethylene Glycol Phenyl Ether, and 2-Phenoxyethanol.
A microparticle gate was established in agreement with the published method for characterization of circulating cell-derived microparticles by preliminary standardization experiments using a blend of size-calibrated 0.3, 0.5, 0.9, and 3 μm fluorescent beads, Megamix®, Biocytex, Marseille, France [27 (link), 28 (link)]. To enlarge the scale of fluorescent beads, in some experiments, Trucount beads (Beckton Dickinson) of 10 μm were added.
A gate for DNA-anti-DNA immune complexes for each series of experiments was built around DNA-anti-DNA immune complexes observed in the aliquot of the pool of 15 SLE serum samples incubated with calf thymus DNA. See below for more details and for compensations.
8
Flow Cytometric Analysis of Platelet Microparticles
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Analyses of labeled samples were performed on a Cytomics FC500 flow-cytometer (Beckman Coulter) as previously described (Robert et al., 2009 (link)). Briefly, after standardization of the protocol with a blend of monodisperse fluorescent beads of three diameters (0.5, 0.9 and 3 μm, Megamix, Stago, Biocytex, Marseille, France), optimal instrument settings and the MP region were defined. Megamix beads were run before starting each analysis in order to control and, eventually, to adjust FCM-settings. Forward (FS) and side (SS) scatter parameters were plotted on logarithmic scales to best cover a wide size range. PMPs were gated in the MP window and defined as single CD61 (or CD41)+ events or dual-positive phosphatidylserine (PS)+/CD61 (or CD41)+ events, as seen in dual-color fluorescence plots after staining with annexin V-FITC and CD61 (or CD41)-PE. Single staining controls were used to check fluorescence compensation settings and to set up positive regions. Each tube was run for 1 min at medium flow-rate, with a maximum delay of 30 min after the end of staining.
To limit background noise from dust and crystals, flow cytometric analyses were performed using a 0.22 μm-filtered sheath fluid (IsoflowTM, Beckman Coulter). CXP ACQUISITION and CXP ANALYSIS software packages (Beckman Coulter) were used for data acquisition and analysis, respectively.
To limit background noise from dust and crystals, flow cytometric analyses were performed using a 0.22 μm-filtered sheath fluid (IsoflowTM, Beckman Coulter). CXP ACQUISITION and CXP ANALYSIS software packages (Beckman Coulter) were used for data acquisition and analysis, respectively.
9
Extracellular Vesicle Antigen Analysis
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PPP EV membrane antigen levels were assessed by flow cytometry (CytoFLEX, Beckman Coulter, United States) using fluorescent antibodies (Supplementary Table S1 ) according to the MIFlowCyt-EVs standards (Welsh et al., 2020 (link)). Specifically, EV gates were set using Megamix, a mix of fluorescent beads (0.5/0.9/3 µm beads; Biocytex, Marseille, France) (Supplementary Figure S4A ), a “gold standard” size gate calibration bead mix (Robert et al., 2009 (link); Robert et al., 2012 (link)) and 0.2 µm polystyrene beads (Malvern Pananalytical, United Kingdom) (Supplementary Figure S4B ). EV analysis included several controls that were used to set the analysis including buffer only, unstained samples, appropriate isotype controls and single-stain antibodies as required (Supplementary methods MIFlowCyt checklist) (Lee et al., 2008 (link)). Events were collected by time at a flow rate of 10 µL per minute. Controls and samples were analyzed in the same acquisition setting and reagent conditions. Instrument configuration and settings: Gain: FSC 500; SSC 100; Violet SSC 40; PE 120; APC400; FITC 100, Threshold: manual 10000 height.
10
Characterization of Microvesicle Composition
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Composition and cell origin of MVs was done as described [35 (link), 36 (link)]. Briefly, MVs were permeabilized with Fix/Perm buffer (Biolegend), incubated with Fc blocking buffer (Biolegend), and labeled using antibodies to HMGB1, GFAP (astrocytes), Na+/K+ ATPase α3 (neurons), and CD11b (microglia). Samples are incubated with fluorescent secondary antibodies when appropriate. The Stratedigm S1000Ex was used to assess the stained MVs at the UNC Flow Cytometry Core Facility. Size gates to identify MVs (0.1–1.0 μm) were set using MegaMixTM (BioCytex) size gating beads (Additional file 1 : Figure S1A). Single color controls for each primary antibody, compared to unstained media, were used to develop the compensation matrix and distinguish background staining from specific staining using FloJoTM software version 10.0 (Additional file 1 : Figure S1B). Approximately 5% of the MVs stained positive for lactadherin, which binds phosphotidyl-serine (PS). Of the PS+ MVs, ethanol increased HMGB1 in a dose-dependent fashion up to 123% of controls at 75 mM (not shown).
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