Multiplex imaging of murine bone marrow using Phenocycler 2.0™

Bone marrow (BM) is a tissue that is of great importance to several areas of basic and translational research, including hematology,oncology, bone biology, and immunology. It is unique in that it is gelatinous in nature but housed in a hard casing of bone.Traditionally, flow cytometry and immunofluorescence (IF) techniques have been employed to study the composition of cellularinteractions and elements of the BM. However, it has been challenging to study the BM in an unperturbed state using multiplefluorescent probes at a time to fully appreciate the diverse cell populations and their interactions and relative positioning with eachother. This protocol addresses how Phenocycler 2.0 TM, which uses co-detection by indexing (CODEX) in conjunction with HALO4.0TM image analysis software, can overcome the obstacles faced by traditional techniques used to study the BM in an unperturbedstate.