Postnatal-dissociated cortical neuron cultures were prepared as previously described (31 , 36 (link)), from newborn B6 mice. Neurons were plated into 35 mm glass bottom Petri dishes at a 1 million/ml density in culture medium consisting of Basal Medium Eagle supplemented with 10% bovine calf serum and 1% penicillin–streptomycin. On day in vitro 2 (DIV2), culture medium was changed to Neurobasal A medium, supplemented with B27-plus reagent (Invitrogen), Glutamax, and 1% penicillin–streptomycin. Neurons were transfected with the CryBAR optogenetic system (6 μg plasmid/plate) on DIV5 using Lipofectamine LTX reagent. On DIV7, culture medium was removed and neurons were placed in imaging solution (Mg-free HEPES-buffered artificial cerebrospinal fluid (37 (link))) Live-cell imaging was performed before and after illumination using a Leica DMi8 Live Cell Imaging System. Animal use protocols were approved by East Carolina University Institutional Animal Care and Use Committee.
Dmi8 live cell imaging system
Manufactured by Leica
The DMi8 Live Cell Imaging System is a high-performance microscope designed for live-cell imaging. It features advanced optical components and a modular design to provide researchers with a versatile platform for conducting a wide range of live-cell experiments.
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Lab products found in correlation
Dfc900 gt camera, by Leica (4 mentions)
B27 plus reagent, by Thermo Fisher Scientific (2 mentions)
Hcx pl apo 63 1.40 to 0 60 numerical objective oil objective, by Lumencor (2 mentions)
Hcx pl apo 63x 1.40 0 60na oil objective, by Lumencor (2 mentions)
Led light engine, by Leica (2 mentions)
Neurobasal a medium, by Thermo Fisher Scientific (1 mentions)
Lsm 700 laser scanning microscope, by ZenBio (1 mentions)
Lsm 700 laser scanning microscope, by Zeiss (1 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions)
6 protocols using dmi8 live cell imaging system
1
Optogenetic Stimulation of Cortical Neurons
2
Live Cell Imaging with Leica DMi8 System
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A Leica DMi8 Live Cell Imaging System, equipped with an OKOLab stage-top live cell incubation system, LASX software, Leica HCX PL APO 63×/1.40 to 0.60 numerical objective oil objective, Lumencor LED light engine, CTRadvanced+ power supply, and a Leica DFC900 GT camera, was used to acquire images. Exposure times were set at 200 ms (mCherry, 553 nm) and 50 ms (GFP, 480 nm), with LED light sources at 50% power, and images were acquired every 30 s over specified time course.
3
Live Cell Imaging of Fluorescent Proteins
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A Leica DMi8 Live Cell Imaging System, equipped with an OKOLab stage-top live cell incubation system, LASX software, Leica HCX PL APO 63x/1.40–0.60na oil objective, Lumencor LED light engine, CTRadvanced+ power supply, and a Leica DFC900 GT camera, was used to acquire images. Exposure times were set at 50 ms (GFP, 470 nm) and 200 ms (mCherry, 550 nm), with LED light sources at 50% power, and images acquired every 30 seconds over a 10 min time course. TIRF imaging was conducted using a 100X TIRF compatible objective and 10% laser power.
4
Live-Cell Imaging of Cellular Signaling
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HEK293T cells and Neuro2a cells were imaged on a Leica DMi8 Live Cell Imaging System, equipped with an OKOLab stage-top live cell incubation system, LASX software, Leica HCX PL APO 63×/1.40 to 0.60 numerical objective oil objective, Lumencor LED light engine, CTRadvanced+ power supply, and a Leica DFC900 GT camera. The mCherry channel (553 nm, 50% power, 200 ms exposure) was used for pre, during, and post-light visualization. GFP channel (480 nm, 50% power, 50 ms exposure) was used for Cry2 activation. Images were captured in 30 sec intervals.
Live-cell confocal images of HEK293T cells were collected on Zeiss LSM 700 laser scanning microscope equipped with a stage-top incubator and ZEN Black 2012 software. Images were captured on the mCherry channel (553 nm) and GFP channel (480 nm) for blue light illumination in 30 sec intervals. For ERK-KTR cell experiments, ERK-KTR was visualized on the GFP channel (5% power), and the nuclear stain was visualized on the DAPI channel (395 nm, 5% power). All images were analyzed using Fiji Image J software.
Live-cell confocal images of HEK293T cells were collected on Zeiss LSM 700 laser scanning microscope equipped with a stage-top incubator and ZEN Black 2012 software. Images were captured on the mCherry channel (553 nm) and GFP channel (480 nm) for blue light illumination in 30 sec intervals. For ERK-KTR cell experiments, ERK-KTR was visualized on the GFP channel (5% power), and the nuclear stain was visualized on the DAPI channel (395 nm, 5% power). All images were analyzed using Fiji Image J software.
5
Confocal and Widefield Microscopy of Cells
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Confocal Microscopy: Confocal images of fixed cells were collected with a Zeiss LSM 700 laser scanning microscope using ZEN Black 2012 software. Fluorescence images were colorized and overlaid using FIJI software.
Widefield Microscopy: A Leica DMi8 Live Cell Imaging System, equipped with an OKOLab stagetop live cell incubation system, LASX software, Leica HCX PL APO 63x/1.40-0.60na oil objective, Lumencor LED light engine, CTRadvanced+ power supply, and a Leica DFC900 GT camera, was used to acquire images. Exposure times were set at 200 ms (mCherry, 550 nm) and 50 ms (GFP, 480 nm), with LED light sources at 50% power, and images acquired every 30 seconds over specified time course.
Widefield Microscopy: A Leica DMi8 Live Cell Imaging System, equipped with an OKOLab stagetop live cell incubation system, LASX software, Leica HCX PL APO 63x/1.40-0.60na oil objective, Lumencor LED light engine, CTRadvanced+ power supply, and a Leica DFC900 GT camera, was used to acquire images. Exposure times were set at 200 ms (mCherry, 550 nm) and 50 ms (GFP, 480 nm), with LED light sources at 50% power, and images acquired every 30 seconds over specified time course.
6
Optogenetic Stimulation of Postnatal Cortical Neurons
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Postnatal dissociated cortical neuron cultures were prepared as previously described (Bunner, Dodson, Szatmari, & Hughes, 2021; Chang et al., 2017) (link), from newborn B6 mice. Neurons were plated into 35 mm glass bottom Petri dishes at a 1 million/ml density in culture medium consisting of BME supplemented with 10% BCS and 1% Penicillin-Streptomycin. On day in vitro 2 (DIV2), culture medium was changed to Neurobasal A medium, supplemented with B27-plus reagent (Invitrogen), Glutamax and 1% Penicillin-Streptomycin. Neurons were transfected with the CryBAR optogenetic system (6 µg plasmid/plate) on DIV5 using Lipofectamine LTX reagent (Invitrogen). On DIV7, culture medium was removed and neurons were placed in imaging solution (Mg-free HEPES buffered aCSF (Sun, Smirnov, Kamasawa, & Yasuda, 2021 (link))) Live cell imaging was performed before and after illumination using a Leica DMi8 Live Cell Imaging System.
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