Single-molecule localization microscopy reveals the molecular organization of endogenous membrane receptors

Super-resolution microscopy in combination with genetic labeling methods
allows imaging of single proteins in cells. However, visualizing
endogenous proteins on primary cells remains challenging due to the use of
sterically demanding antibodies for labeling. Here, we demonstrate how
immunolabeling conditions and antibody crosslinking influence the
quantification and identification of membrane receptor stoichiometry on
cells using single molecule localization microscopy. We developed an
optimized immunolabeling and analysis protocol and demonstrate the
performance of the approach by resolving the molecular organization of
endogenous CD45, CD69, and CD38 on Jurkat T cells. To demonstrate the
usefulness of the method for immunotherapy applications, we investigated
the interaction of primary multiple myeloma cells with the therapeutic
monoclonal antibodies (mAbs) daratumumab and isatuximab, and a polyclonal
anti-CD38 antibody. Our approach might lay the foundation for improved
personalized diagnostics and treatment with therapeutic antibodies.