urinary EVs were isolated using StemCell tech. magnetic bead kit for total exosome isolation, trying to run few antibody assays through flowcytometry. that kit cannot be used with NanoSight, therefore I'm asking on how to run them through flowcytometry and determining their number prior to the flow assay?
Samiullah Khan
Prepare your samples: Thaw the EV samples and dilute them to an appropriate concentration for the assay. You may need to optimize the dilution factor for your specific assay and sample type. Coat your assay plates: Coat your assay plates with a capture antibody that is specific for the EV marker(s) you are interested in detecting. Incubate the plates overnight at 4°C to allow the antibodies to bind to the plate surface. Block non-specific binding sites: Block non-specific binding sites on the assay plates with a blocking buffer, such as BSA or milk, for 1-2 hours at room temperature. Add your EV samples to the assay plates: Add your diluted EV samples to the assay plates and incubate for 1-2 hours at room temperature or overnight at 4°C. This will allow the EVs to bind to the capture antibodies on the plate surface. Wash the assay plates: Wash the assay plates to remove any unbound EVs or other contaminants using an appropriate washing buffer. Repeat this step several times to ensure the removal of all unbound material. Add detection antibodies: Add a detection antibody that is specific for your target EV marker(s) to the assay plates and incubate for 1-2 hours at room temperature. The detection antibody should be labeled with a detectable signal, such as a fluorescent or chemiluminescent tag. Wash the assay plates again: Wash the assay plates again to remove any unbound detection antibodies. Read the assay results: Measure the signal generated by the detection antibodies on the assay plates using a suitable detection method, such as a plate reader. You can use the signal generated by the detection antibodies to quantify the amount of EVs present in your samples.
To run an antibody assay for urinary extracellular vesicles (EVs) isolated through the magnetic bead method, you can follow these general steps:
It is important to note that the specific details of the antibody assay may vary depending on the specific EV markers and antibodies used. Optimization of the assay conditions may also be necessary for your specific experimental setup.
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