DiBAC4(5) is a translational MP dyes that redistributes within the cell membrane as MP changes. MP depolarization results in an increase in fluorescence intensity of cells at Ex/Em = 590/616nm. Conversely, hyperpolarization is indicated by a decrease in fluorescence. PMA-differentiated THP-1 cells were cultured in 35mm imaging dish (ibidi) with a concentration of 4×105 cells/ml. Cells were stained in 200 nM DiBAC4(5) in 5mM K+ buffer at 37°C for 30 min. Then the staining buffer was removed without washing and crystals (silica, Al(OH)3, CPPD) or inhibitors (glyburide or CFTRinh-172) in fresh 5mM K+ buffer containing 200nM DiBAC4(5) was added to the cells. For high K+ condition, staining buffer was replaced with 100mM K+ buffer containing 200nM DiBAC4(5). We captured 5 different fields at each time-point on fluorescent microscope, and analyzed the fluorescence intensity of all the cells by ImageJ. The relative fluorescence intensity indicates MP changes under different condition, which is normalized with the mean fluorescence intensity of non-treated cells (Laskey et al., 1992 (link)).
35 mm imaging dish
Manufactured by Ibidi
The 35 mm imaging dish is a laboratory equipment designed for cell culture and microscopic observation. It provides a sterile and controlled environment for growing and examining cells. The dish has a diameter of 35 mm and is made of high-quality materials to ensure durability and optimal performance.
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6 protocols using 35 mm imaging dish
1
Membrane Potential Imaging Assay
2
Measuring Calpain Activity in Immune Cells
3
Membrane Potential Imaging Assay
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DiBAC4(5) is a translational MP dyes that redistributes within the cell membrane as MP changes. MP depolarization results in an increase in fluorescence intensity of cells at Ex/Em = 590/616nm. Conversely, hyperpolarization is indicated by a decrease in fluorescence. PMA-differentiated THP-1 cells were cultured in 35mm imaging dish (ibidi) with a concentration of 4×105 cells/ml. Cells were stained in 200 nM DiBAC4(5) in 5mM K+ buffer at 37°C for 30 min. Then the staining buffer was removed without washing and crystals (silica, Al(OH)3, CPPD) or inhibitors (glyburide or CFTRinh-172) in fresh 5mM K+ buffer containing 200nM DiBAC4(5) was added to the cells. For high K+ condition, staining buffer was replaced with 100mM K+ buffer containing 200nM DiBAC4(5). We captured 5 different fields at each time-point on fluorescent microscope, and analyzed the fluorescence intensity of all the cells by ImageJ. The relative fluorescence intensity indicates MP changes under different condition, which is normalized with the mean fluorescence intensity of non-treated cells (Laskey et al., 1992 (link)).
4
Measuring Calpain Activity in Immune Cells
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THP-1 cells or BMDCs were plated in 35 mm imaging dish (ibidi). Cells were pretreated with 3 mM or 100 mM K+ buffer, or medium with or without glyburide (200 μM) for 30 min in cell culture incubator. Cells were then moved to fluorescent microscopy with temperature control at 37°C. Half of the medium in dish was pipetted up to suspend calpain substrate BOC-LM-CMAC (50 μM) together with or without silica and then added back. After cleavage by calpain, the product emits blue-fluorescence with excitation/emission maxima ~351/430 nm. 30 min later, images were captured and CMAC fluorescence intensity in each cell was analyzed by ImageJ. For measuring calpain activity in cells under voltage control by patch clamp, the cell was patched first, CMAC was added, and then the MP was changed to hold at −40 mV, 0 mV or −100 mV. Pictures were taken at 10 min, 20 min and 30 min. CMAC fluorescence intensity in the patched cell was normalized to other unpatched cells (resting MP) in the same field.
5
Mitochondrial Membrane Potential in Krill Oil Treated Fibroblasts
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A TMRE (Tetra methyl rhodamine, ethyl ester) Mitochondrial Membrane Potential assay kit was used to measure the mitochondrial membrane potential. In total, (late passaged) 50,000 BJ fibroblasts cells were plated onto 35 mm imaging dish (IBIDI) with polymer coverslip bottom. 24 hours post seeding cells were treated with 100 μg/ml krill oil and the cells were allowed to grow for another 5 days at 37°C in 5% CO2 incubator. On day 6, TMRE assay was performed according to the manufacturer's protocol. Briefly, cells were stained at a final concentration of 200 nM TMRE and incubated in dark for 30 min at 37°C. After incubation, cells were washed again with PBS and live cell imaging was done on Zeiss LSM780 confocal microscope, at ×63 magnification using appropriate filters.
6
Immunofluorescence Staining Protocol
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Cells were seeded in 35 mm imaging dish (ibidi), and fixed with 4% formaldehyde on ice for 15 min and permeabilized in 0.3% Triton X-100 (in PBS) for 10 min. Blocking for an hour and cells stained with indicated antibody for 16 h at 4 °C. Alexa Fluor 488 dye (Thermo Fisher Scientific) was used as secondary antibody for green-fluorescent dye. The cell nuclei were stained by DAPI. The image of fluorescence was taken by Leica TCS SP5 AOBS Confocal Spectral Microscopy using a 63x oil-immersion objective len and a 10x eyepiece. A scale bar showing 25 μm was arranged at the lower right part of each image.
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