SKU: CBS11
vTAG™ ANTI-HUMAN CD63 ANTIBODY
NO WASH, QUANTITATIVE CD63 MEASUREMENT BY vFC™

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Conjugate(s): PE
Est. Limit of Detection: 30 Molecules per EV

 

Cargo analysis using vTag™ antibodies should be accompanied by a vesicle specific analysis method such as vFC™. vTag™ antibodies bundled with the vFC™ assay will be discounted 25%.

Certificate of Analysis

TDS MSDS

$105.00$275.00

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Tested Reactivity: Anti-Human.
Other Published Reactivity: African Green, Baboon, Cynomolgus, and Rhesus.
Conjugate: PE (see dropdown at the top for available options).
Sensitivity: Limit of detection is estimated at 30 molecules per vesicle in vFC™ for the PE conjugate. Change selection above for LOD of the antibody conjugated to a different fluorophore. LOD is estimated using antibody binding beads and will vary depending upon sample, instrument, and other experimental conditions.
Application: Optimized for vFC™ with no wash steps required.
Clonality: Monoclonal.
Clone: H5C6.
Host and Isotype: Mouse IgG1, kappa.
Positive Control Sample: EVs from human cell lines, especially U87MG or MCF7.
Localization of Staining: Vesicle Surface.
Buffer and Stabilizer: 10mM PBS with 0.05% BSA & 0.05% azide.
Concentration: 5 µl/test.
Purification Method: Affinity purified during antibody production, additionally purified during conjugation to remove free dye and unbound antibody.
Immunogen: T cell line HPB-ALL.
Storage Conditions: Store undiluted between 2°C and 8°C. Protected from prolonged exposure to light. Do not freeze. Stable for 1 year when properly stored.

CD63 Immunostaining in vFC™?

This protocol provides a standardized means of analyzing individual vesicles to measure size, concentration, and CD63 expression simultaneously via vFC™. vFC™ (#CBS4) is an assay that enables specific detection and characterization of individual extracellular vesicles such as exosomes and microvesicles on commercially available flow cytometers. Because vFC™ is vesicle specific, we can characterize vesicles and cargo in a highly-reproducible, no-wash assay without artifacts from debris or reagent aggregates. In this protocol, vesicles will be stained using our vTag™ Anti-Human CD63 Antibody. See protocol 2 in the vFC™ kit handbook for additional considerations and full protocol details.

Procedure

  1. Dilute sample containing EVs to approximately 5×106 extracellular vesicles per µl. To determine vesicle concentration for an unknown sample, see supporting protocols included in the vFC™ kit.
  2. Prepare a sufficient amount of vFluor™ Red for all samples and controls (5 µl per well) according to kit instructions
  3. In duplicate, in the included 96 well plate, add 35 µl of dilution buffer, 5µl of prepared vFluor™ Red, 5 µl of vTag™ anti-human CD63 antibody, and 5 µl of EV samples.
  4. Incubate in the dark at room temperature for 1 hr.
  5. Perform serial dilution of the samples as described in the assay handbook. Briefly, samples are to be diluted from between 1:100 to 1:1000 from staining concentration depending upon the flow cytometer being used.
  6. Run the diluted sample of labeled EVs on a qualified flow cytometer.

CD63 is a member of the tetraspanin family. In cells, tetraspanins are important to many biological functions including cell adhesion and motility as well as signaling and protein trafficking.

CD63 predominantly associates with the outer membrane of MVBs in cells leading researchers to originally describe it as a universal marker for vesicles originating within cells, principally exosomes. Recent evidence, of course, suggests that there are populations of exosomes and other vesicles that do not express measurable amounts of CD63. More information on CD63 coming soon!

vFC™ VESICLE FLOW CYTOMETRY EV ANALYSIS ASSAY
FOR COUNTING AND SIZING VESICLES IN BIOFLUIDS, MEDIA, OR BUFFER

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Summary

Sample Format:Vesicles from cells in culture
Vesicle Size:>70nm
Certificate of Analysis

Protocol Handbook MSDS
$450.00$2,050.00
Size

20 Tests, 100 Tests

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NO WASH, QUANTITATIVE CD63 MEASUREMENT BY vFC™