Simultaneous Monitoring of ER, Mitochondrial, and Cytosolic Calcium Levels

To monitor ER [Ca²⁺] ([Ca²⁺]_ER) or mitochondrial [Ca²⁺] ([Ca²⁺]_MITO) simultaneously with cytosolic [Ca²⁺]_i, mpkCCD cells were transfected with either ER Ca²⁺ biosensor R-CEPIA1er (Addgene Plasmid #58216, λ_ex: 543 nm, λ_em: 560–600 nm) or mitochondrial Ca²⁺ biosensor mito-RCaMP1h (Addgene Plasmid #105013, λ_ex: 543 nm, λ_em: 560–600 nm) (Suzuki et al., 2014 (link)). Cells were seeded at 6 × 10⁴ cells/cm² on collagen coated glass bottom dishes for 24 h before transfection. Cells were transfected with Lipofectamine (0.5 µg DNA/1 × 10⁵ cells) for 24 h according to manufacturer’s instruction. Studies were performed in transfected cells from 48 to 72 h after transfection. Cytosolic [Ca²⁺]_i was monitored simultaneously with cell permeant Ca²⁺ sensitive fluorescence probe (Cal-520/AM) in the transfected cells incubating with phenol red free medium (1:1 mixture of DMEM/Ham’s F12 medium, Gibco). To monitor ER-mitochondrial Ca²⁺ transfer in mpkCCD cells, cells were co-transfected with the ER Ca²⁺ biosensor (G-CEPIA1er, Addgene Plasmid #58215, λ_ex: 488 nm, λ_em: 510–540 nm) and mitochondrial biosensor mito-RCaMP1h. Fluorescent images were collected with the respective laser lines for excitation and spectral windows for emission using the Lecia TCS SP5 imaging system.