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For detecting exosomal protein markers by flow cytometry, are beads necessary? Are there specific steps?
Flow cytometry can detect exosomal marker proteins. Specific steps are as follows:
(1) Process the sample and extract exosomes.
(2) Antibody incubation: Resuspend the extracted exosomes in PBS containing 2% BSA. Perform primary and secondary antibody incubations sequentially to obtain the CD63-labeled exosome detection solution.
(3) Set flow cytometer parameters: Run the flow cytometer normally and set the parameters.
(4) Detection: Resuspend the CD63-labeled exosome detection solution in PBS and load it for detection.
(5) Use flow cytometry analysis software to analyze the data and obtain quantitative detection results for exosomes.
For exosome-related proteins like CD81, CD9, CD63, and calnexin, can they not be stored after boiling? I heard that because these are membrane proteins, they cannot be boiled? Other markers like HSP70 and TSG101 worked fine. Is this true?
Membrane proteins are generally not recommended for boiling. Usually, after boiling the sample, the bands appear very smeared and cannot be recognized well by the antibody. This might be because some antibodies recognize conformational (3D) epitopes rather than linear epitopes.