SKU: CBS12
vTAG™ ANTI-HUMAN CD81 ANTIBODY
NO WASH, QUANTITATIVE FLAG MEASUREMENT BY vFC™
(Copy)

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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

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Tested Reactivity: Routinely tested against human CD81.
Additional Reactivity: This clone also reacts with CD81 from non-human primates including african green, cynomolgous, and rhesus monkeys as well as baboons and may be suitable for labeling vesicles from these species.
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: 5A6.
Host and Isotype: Mouse IgG1&kappa.
Positive Control Sample: EVs produced in HEK293T cells.
Localization of Staining: Vesicle Surface.
Buffer and Stabilizer: 10mM PBS with 0.05% BSA & 0.09% azide.
Concentration: 5 µl/test.
Purification Method: Affinity purified during antibody production, additionally purified during conjugation to remove free dye and unbound antibody.
Immunogen: Human OCI-LY8 cell line.
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.

CD81 Immunostaining in vFC™?

This protocol provides a standardized means of analyzing size, concentration, and CD81 in EVs from human and non-human primate samples. This protocol utilizes vFC™ (#CBS4), 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-CD81 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-CD81 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.

CD81 is another tetraspanin characteristically found on vesicles (exosomes, microvesicles, etc.).  Once considered one of the universal EV marker, we now know that CD81 is one of dozens of tetraspanins expressed in unique patterns characteristic of vesicles from particular cell types.  CD81 is expressed at high concentrations on EVs from HEK293T cells, and is also expressed on some vesicles from macrophage or monocyte derived cell lines.  It is either absent, or expressed below the limit of detection on extracellular vesicles derived from platelets or red blood cells.

CD81 generally functions in regulation of  cell development, activation, growth and motility and may be of interest in functional studies investigating EV cargo.

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

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Summary

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

Protocol Handbook MSDS
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vCAL™ ANTIBODY CAPTURE NANOBEADS

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NO WASH, QUANTITATIVE FLAG MEASUREMENT BY vFC™
(Copy)”

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