vTAG™ ANTI-HUMAN CD235AB ANTIBODY
NO WASH, QUANTITATIVE CD235AB MEASUREMENT BY vFC™

This protocol is a summary of steps required to quantify CD235ab on EVs via vFC™. A full version of this protocol is described in protocol 2 in the vFC™ (#CBS4) handbook and should be used for actual lab work as it contains necessary instrument calibration steps, data analysis protocols, and other information beyond the scope of this overview. vFC™ 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-CD235ab antibody.

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-CD235 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.