Subcellular Euclidean distance measurements with multicolor fluorescence localization imaging in cultured cells

This protocol measures the 3D Euclidean distance (Δ_3D) between two/three fluorescently labeled kinetochore components in fixed samples using Kinetochore Delta software (KiDv1.0.1, MATLAB based). Overestimation of mean Δ_3D is corrected through a Bayesian algorithm, with Δ_EC distances reflecting the ensemble average positions of fluorophores within a kinetochore population. This package also enables kinetochore categorization, which can be used to sub-sample kinetochores and measure Δ_EC. Together, this allows the dynamic architecture of human kinetochores to be investigated (tested in hTERT-RPE1 cells). For complete details on the use and execution of this protocol, please refer to Roscioli et al. (2020).