A Cell-Based Protocol to Assess Manganese Content and Relative Transport Activity of Manganese Transporters

Manganese (Mn) is an essential trace element whose intracellular homeostasis is tightly controlled by specialized membrane transporters. Dysregulation of Mn transport leads to pathological Mn accumulation and severe human disease; however, efficient and quantitative cell-based methods for assessing Mn²⁺ transporter activity remain limited. Here, we present an optimized cellular Fura-2 manganese extraction assay (CFMEA) that enables robust quantification of cellular Mn content and provides a normalized framework for assessing relative Mn²⁺ transport activity in a high-throughput format. This protocol integrates Fura-2-based fluorescence detection of Mn²⁺ at the Ca²⁺ isosbestic excitation wavelength with dsDNA quantification to normalize dsDNA levels in cell extracts and immunoblotting to account for transporter protein expression levels. Cells expressing Mn²⁺ transporters are exposed to MnCl₂ in 96-well plates, washed to remove extracellular Mn²⁺, and lysed in a Fura-2-containing extraction buffer. Fluorescence quenched by Mn²⁺ is quantified and converted to cellular Mn content using a cell-free Mn-Fura-2 standard curve and then normalized to dsDNA content and protein abundance to determine relative transporter activity. This workflow provides a relatively sensitive, reproducible, and low-cost approach for comparative analysis of Mn²⁺ transporters and their variants across multiple cell types. The protocol is demonstrated using the Mn²⁺ efflux transporter SLC30A10 in HEK293T cells and is readily adaptable for studying other Mn²⁺ transport pathways. Key features • High-throughput, cell-based assay for quantifying cellular manganese content and assessing relative Mn²⁺ transporter function. • Enhanced accuracy and reproducibility by integrating double-stranded DNA quantification and protein normalization into the cellular Fura-2 manganese extraction assay (CFMEA) workflow. • Workflow compatible with diverse cell types and Mn²⁺ transporters, including systems overexpressing SLC30A10 in HEK293Tcells.