vFC™ VESICLE FLOW CYTOMETRY EV ANALYSIS ASSAY
FOR COUNTING AND SIZING VESICLES IN BIOFLUIDS, MEDIA, OR BUFFER
$450.00 – $2,100.00
Description
Sample Type: Suitable for characterization of extracellular vesicles directly from cell culture supernatant or biofluids such as plasma, urine, or CSF. Can also be used to analyze EVs separated from complex samples using various techniques. See kit manual for sample type specific assay considerations.
Species: Selected above. Human and mouse kits contain an antibody to label common EV surface proteins and an appropriate positive control. Although most reagents are not species specific, certain controls restrict the use of this kit to human samples.
Limits of Detection: vFC™ is suitable for the enumeration of vesicles down to 50nm from background. By following our staining protocols and using vTag™ monoclonal antibodies, surface antigen detection down to 10 molecules per vesicle is possible. Limit of detection is instrument specific. See product manual for additional information.
Cargo Measurement: vFC™ enables quantitative cargo measurement in a no-wash format using fluorescently labeled antibodies. We recommend vTag™ antibodies which perform optimally and have been appropriately titrated for vFC™.
Instrument Requirements: vFC™ can be run on sensitive, commercially available flow cytometers including the Beckman Coulter CytoFLEX®, the Luminex CellStream® and ImageStream®, and the Cytek® Aurora.
Sample Storage: Care should be taken to keep assay and storage conditions consistent and report all procedures. If vesicles cannot be analyzed fresh, we recommend storing at -80°C and aliquoting to avoid unnecessary freeze/thaw cycles.
Kit Storage: CAUTION! Multiple storage temperatures required. See product manual for instructions.
What are the limitations of the assay?
vFC™ assay (#CBS4) is intended for the analysis of vesicles from cell culture in supernatant or buffer. It is also useful for analyzing vesicles separated from biofluids. It can detect vesicles down to 50nm on currently available instrumentation. Limit of detection of surface markers per vesicle is approximately 10 molecules (depending on the instrument and the vTag™ antibody used). We have characterized limits for several instruments. Please contact us for instrument specific sensitivity assessments.
Why am I not seeing events from the Lipo100™ control sample?
There are several possible reasons for this issue. First, ensure that the instrument you’re using is suitable for vFC™. Next, make sure that the instrument has been properly set up and qualified. For instrument setup procedures see the instrument setup and configuration document under the datasheets and documentation tab. Finally, double check that samples are being properly diluted. Pay careful attention to details such as the plate being used for dilutions as certain plastics may bind vesicle and significantly impact EV counts. For additional support, chat with us by clicking on the icon at the bottom right of the window.
Why am I seeing high background events?
High number of background events can arise from multiple sources. Ensure proper dilution of samples and only run vesicles that are appropriate for this kit. Also, rarely carryover from samples may lead to background which is especially noticeable in blank samples. This is most commonly observed after running small beads. Run a daily clean cycle prior to continuing analysis. If issues continue, run beads diluted 1:10 in cleaning solution in the future.
CellStream® Protocols
CytoFLEX® Protocols
Cytek® Aurora Protocols
ImageStream® Protocols
Kit Overview
If this is your first time running vFC™, start with the appropriate instrument setup protocol for your flow cytometer and then complete protocol 0.1 before moving into analysis of your samples. This will ensure your instrument is performing well and will provide rough calibration of data to enable initial interpretation. Protocol 0.1 will also require vCal™ NANORAINBOW BEADS. Next, for all new samples, always run protocol 1. Protocol 1 provides essential controls to demonstrate EV specificity and singly EV resolution of the assay. Last, and for routine measurement of samples, run protocol 2 to determine the size distribution of EVs and to quantify EV concentration and copy number of various surface markers. Prior to publication and to ensure accuracy of the data from the assay run protocols 0.2-0.4 and apply the instrument specific calibrations to your data as outlined in the protocols.
FCS Express and FCS Express Reader Analysis Layouts
CytoFLEX®
CellStream®
Cytek® Aurora
Sample Data
CytoFLEX®
CellStream®
- Welsh, J. A. et al. MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments. J. Extracell. Vesicles 9, (2020).
- Sandau US, et al. Methamphetamine use alters human plasma extracellular vesicles and their microRNA cargo: An exploratory study. Journal of extracellular vesicles. 2020;10(1):e12028.
- Shpigelman J, et al. Generation and Application of a Reporter Cell Line for the Quantitative Screen of Extracellular Vesicle Release. Frontiers in pharmacology. 2021;12:840.
- Crooks ET, et al. Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens. PLoS pathogens. 2021;17(10):e1009807.
- Oh C-k, et al. S-Nitrosylation of p62 Inhibits Autophagic Flux to Promote α-Synuclein Secretion and Spread in Parkinson’s Disease and Lewy Body Dementia. The Journal of Neuroscience. 2022:JN-RM-1508-21. doi: 10.1523/jneurosci.1508-21.2022.
- Yan W, et al. Cancer-cell-secreted miR-122 suppresses O-GlcNAcylation to promote skeletal muscle proteolysis. Nature Cell Biology. 2022. doi: 10.1038/s41556-022-00893-0.
Additional information
| Size | 96 Wells, 5 x 96 Wells |
|---|---|
| EV Marker (Antibody) ⓘ | Anti-Human TS Cocktail (CD9, CD63, CD81) [PE], Anti-Human TS Cocktail (CD9, CD63, CD81) [PE-Cy7], Anti-Mouse TS Cocktail (CD9, CD63, CD81) [PE], Anti-Mouse TS Cocktail (CD9, CD63, CD81) [PE-Cy7], Other, None |
FOR COUNTING AND SIZING VESICLES IN BIOFLUIDS, MEDIA, OR BUFFER”
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