Measuring ER Calcium Dynamics in INS-1 Cells

INS-1 cells were transfected with GEM-CEPIA1er (Addgene)^{23 (link)} or a ratiometric FRET-based Cameleon probe D1ER²⁴ using Lipofectamine 2000. After 48 h, cells were treated with rotenone or O/A in a Ca²⁺-free KRB buffer (Sigma) for 1 h to inhibit influx of extracellular Ca²⁺ into ER^{34 (link)} or in a culture medium for 1 h. GEM-CEPIA1er fluorescence was measured using an LSM780 confocal microscope (Zeiss) at an excitation wavelength of 405 nm and emission wavelengths of 466 or 520 nm, and F466/F520 was calculated as an index of [Ca²⁺]_ER^{23 (link)}. D1ER fluorescence intensity ratio (F540/F490) was determined using an LSM980 confocal microscope (Zeiss)

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Park K., Lim H., Kim J., Hwang Y., Lee Y.S., Bae S.H., Kim H., Kim H., Kang S.W., Kim J.Y, & Lee M.S. (2022). Lysosomal Ca2+-mediated TFEB activation modulates mitophagy and functional adaptation of pancreatic β-cells to metabolic stress. Nature Communications, 13, 1300.

Publication 2022

GEM-CEPIA1er KRB buffer LSM780 confocal microscope LSM980 confocal microscope

Buffer Cells Confocal microscope Culture medium Fluorescence Fret Inhibit Lipofectamine 2000 Rotenone

Corresponding Organization :

Other organizations : Yonsei University, Chungnam National University, Korea Advanced Institute of Science and Technology, Korea Institute of Brain Science

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Variable analysis

independent variables

Transfection with GEM-CEPIA1er or D1ER probe
Treatment with rotenone or O/A in Ca2+-free KRB buffer or culture medium

dependent variables

GEM-CEPIA1er fluorescence (F466/F520 ratio)
D1ER fluorescence intensity ratio (F540/F490)

control variables

Lipofectamine 2000 for transfection
Ca2+-free KRB buffer (Sigma) or culture medium for treatments
Confocal microscopes (LSM780 and LSM980) for fluorescence measurements

controls

Positive control: Cells transfected with GEM-CEPIA1er or D1ER probe
Negative control: Not explicitly mentioned

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