Protocol for FLOWMIST to profile autoreactivity of mouse monoclonal and serum antibodies by flow cytometry and microscopy

Autoantibodies recognizing surface-bound or intracellular self-antigens are a hallmark of many autoimmune diseases. Here, we present a protocol for FLOWMIST to profile the autoreactivity of mouse monoclonal and serum antibodies using primary mouse splenocytes. We describe steps for antibody staining, quantitative flow cytometric screening, and confocal microscopy to classify antibody-binding patterns. Although demonstrated using mouse splenocytes, the workflow can be readily adapted to different target cells, antibody samples, species, and secondary detection reagents. For complete details on the use and execution of this protocol, please refer to Simpson et al.¹ and Newen et al.².