Cell division and holdfast synthesis timing were observed in live cells on agarose pads by time-lapse microscopy as described previously (Hoffman et al., 2015 (link)) with some modifications. A 1 µl aliquot of exponential-phase cells (OD600 of 0.4 – 0.8) was placed on top of a pad containing 0.8% agarose in M2G or PYE and AF488-WGA (0.5 µg/ml final) sealed under a coverslip with valap. Time-lapse microscopy images were taken every 2 min for 8–12 h using a Nikon Eclipse 90i with a 100 x DIC oil immersion objective, a Nikon 83300 filter cube T-D-F and a Photometrics Cascade K1 camera, or an inverted Nikon Ti-E microscope and a Plan Apo 60X objective, a GFP/DsRed filter cube, and an Andor iXon3 DU885 EM CCD camera. Time-lapse movies were visualized in ImageJ (Schneider et al., 2012 (link)) to manually assess the time of cell division (t0), as t = 0 when a cell newly divides, and the time of holdfast production (tH), as the first frame where the fluorescent lectin signal is detected.
Ixon3 du 885 emccd camera
Manufactured by Oxford Instruments
The IXON3 DU-885 EMCCD camera is a high-performance imaging device designed for scientific applications. It features an electron-multiplying charge-coupled device (EMCCD) sensor that provides high sensitivity and low-light detection capabilities. The camera is capable of capturing images and video with high resolution and low noise levels.
Automatically generated - may contain errors
Lab products found in correlation
Revolution xd system, by Oxford Instruments (3 mentions)
Csu x1, by Yokogawa (2 mentions)
Eclipse 90i, by Nikon (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
Plan apochromat, by Oxford Instruments (1 mentions)
Csu x1 spinning disk, by Yokogawa (1 mentions)
Plan apochromat objective, by Oxford Instruments (1 mentions)
4 protocols using ixon3 du 885 emccd camera
1
Time-Lapse Imaging of Cell Division and Holdfast Formation
2
Imaging Transfected 5-HT6R-EGFP Neurons
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Live neurons transfected with 5-HT6R-EGFP were transferred to the imaging chamber (Live Cell Instrument, Korea), filled with imaging solution (mM: 120 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 15 glucose, 15 HEPES, pH 7.35) or neurobasal media without phenol red (Gibco, Life Technologies) and imaged at 32°C. Temperature was controlled by heating plate base connected to the imaging chamber. Confocal images were acquired using the Revolution XD System (Andor Technology) equipped with Yokogawa CSU-X1 spinning disk confocal unit, 488 nm solid state laser, 561 nm solid state laser, and Andor 6-line laser combiner. Images were obtained using a 60× (NA 1.4) and a 14-bit iXON3 DU-885 EMCCD camera (Andor Technology) through the Metamorph software program (Molecular Device Inc.). For the SB258585 and ST1936 treatment experiments (Figs. 3G–3J ), neurons were maintained at basal states for 6 minutes, then pre-incubated with SB258585 for 20 min, and then treated with ST1936 for 30 min.
3
Neuronal Imaging with Live-Cell Microscopy
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Live neurons grown on the coverslip that were transfected appropriately were transferred to the imaging chamber equipped with heating plate base (Live Cell Instrument, Seoul, Korea); filled with imaging solution (mM: 120 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 15 glucose, 15 HEPES, pH 7.35), and imaged at 32 °C. Confocal images were acquired using the Revolution XD System (Andor Technology) equipped with Yokogawa CSU-X1 spinning disk confocal unit, 488 nm solid state laser, 561 nm solid state laser, 640 nm diode laser, and Andor 6-line laser combiner. Images were taken using a 60x (NA 1.4) or 100x Plan Apochromat objective (NA 1.4) and a 14-bit iXON3 DU-885 EMCCD camera (Andor Technology) using the Metamorph software program (Molecular Device Inc.). We acquired a complete confocal z-sectioning of the region of interest, followed by maximal intensity projection to produce a two-dimensional image using Metamorph.
For glycine stimulation, neurons were treated with 200 μM glycine in Mg2+-free imaging solution with 0.5 μM TTX, 1 μM strychnine, 20 μM bicuculline methiodide for 3−5 minutes. Then, neurons were returned to imaging solution with 0.5 μM TTX, 1 μM strychnine, and 20 μM bicuculline methiodide. Neurons at DIV 15−17 were used for imaging experiments.
For glycine stimulation, neurons were treated with 200 μM glycine in Mg2+-free imaging solution with 0.5 μM TTX, 1 μM strychnine, 20 μM bicuculline methiodide for 3−5 minutes. Then, neurons were returned to imaging solution with 0.5 μM TTX, 1 μM strychnine, and 20 μM bicuculline methiodide. Neurons at DIV 15−17 were used for imaging experiments.
4
Live-Cell Imaging of Receptor Trafficking
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HEK 293 T cells overexpressing SEP-GluA1, SEP-GluA2, SEP-GluN1, or SEP-GluN2A, which had been grown on the coverslip, were transferred to an imaging chamber (Live Cell Instrument, Seoul, Korea) filled with imaging solution (mM: 120 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 15 glucose, 15 HEPES, pH 7.35). The cells were live-imaged confocally every 15 s for 7–8 min before the appropriate drug applications, and then live-cell imaging was continued every 15 s for the next 35 min in the presence of the drugs. Confocal imaging was performed using the Revolution XD System (Andor Technology) equipped with a Yokogawa CSU-X1 spinning disk confocal unit, a 488 nm solid-state laser, a 60× Plan Apochromat objective (NA 1.4), and a 14-bit iXON3 DU-885 EMCCD camera (Andor Technology). Using the Metamorph imaging and analysis software program (Molecular Device Inc.), the complete confocal z-sectioned images were acquired, and maximum intensity projected images were produced.
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