I am conducting a multiplex flow cytometry assay to count the CD3, CD4, CD8 and CD21 markers in bovine PBMCs. However, the manufacturer of CD3 antibody recommends fixing the cells and permeabilizing before staining with the antibodies (the CD3 marker did not work without fixation and permeabilization). On doing so, i have got good results. But i am not convinced with my results because these markers should be extracellular. So, am i producing invalid results by permeabilizing the cells and exposing the intracellular receptors? Any advice would be very helpful. Thanks in advance.
Well, the situation you describe is not necessarily a problem or a error. Some molecules are indeed not suitable for staining after fixation, except for CD3. You need to combine the antibody manual to analyze specifically which domain it binds to. For questions about extracellular and intracellular, you can find relevant experimental protocols on websites such as themofisher.
So my answer is not necessarily.
The permeabilization of PBMCs for flow cytometry analysis can indeed affect the results of the assay and may produce invalid results for extracellular markers. The permeabilization process can allow the antibodies to access intracellular antigens, potentially leading to an over-representation of the marker in question.
For extracellular markers such as CD3, CD4, CD8, and CD21, it is ideal to analyze the markers without permeabilizing the cells, as this allows for the measurement of the markers that are present on the cell surface only. However, if the CD3 antibody requires permeabilization for detection, this may indicate that the CD3 antigen is partially located intracellularly, or that it is associated with intracellular components that need to be accessed for proper detection.
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