SIM was performed using an Applied Precision DeltaVision OMX equipped with a 60× Plan-Apochromat N/1.42 NA objective and processed using softWoRx software (GE Healthcare). Confocal microscopy was performed using either a Leica TCS SP5 laser-scanning confocal microscope (fixed cells) or a Nikon A1R laser-scanning confocal microscope (fixed and live cells). For live-cell imaging of Ls174T-W4 cells, 1 d before imaging, cells were transfected in a T25 flask at 80% confluency using Lipofectamine 2000 as described by the manufacturer (11668; Invitrogen). The next day, cells were plated on glass-bottom dishes and allowed to adhere for 6 h. Imaging was performed on a Nikon A1R confocal microscope equipped with 488- and 561-nm excitation lasers and a 100×/1.49 NA Apo TIRF objective. Doxycycline was added to the media at 1 μg/ml just before image acquisition began. Cells were maintained in a humid environment at 37°C and 5% CO2 using a stage-top incubation system (Tokai Hit). Image acquisition was controlled with Nikon Elements software.
Stage top incubation system
Manufactured by Tokai Hit
The stage-top incubation system is a laboratory equipment designed to maintain a controlled environment for live-cell imaging and microscopy applications. It provides temperature, humidity, and gas regulation to support the growth and observation of cells during experiments.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements software, by Nikon (2 mentions)
Eclipse ti microscope, by Nikon (2 mentions)
Deltavision omx, by Cytiva (1 mentions)
Softworx software, by GE Healthcare (1 mentions)
Elements software, by Nikon (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
A1r laser scanning confocal microscope, by Nikon (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Dna xfect transfection reagent, by Takara Bio (1 mentions)
Fluorobrite dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Orca er ccd camera, by Hamamatsu Photonics (1 mentions)
Zyla 4.2 scmos camera, by Oxford Instruments (1 mentions)
Metamorph 7.5 imaging software, by Molecular Devices (1 mentions)
Orca flash4.0 lt digital cmos camera, by Hamamatsu Photonics (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
4 protocols using stage top incubation system
1
Live-cell imaging of transfected cells
2
Quantitative Enzyme Interaction Analysis
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105 HeLa cells were plated on glass-bottomed 35 mm TC dishes (Cellvis) in 1.5 ml FluoroBrite Dulbecco's modified Eagle's medium (DMEM) (ThermoFisher) supplemented with l-glutamine, sodium pyruvate, and normal (purine-rich) or dialyzed (10 kDa MWCO) FBS (purine-depleted) (Atlanta Biologics). After 48 h, the media was replaced with 1 ml fresh media, and the cells are transfected (Xfect DNA Transfection Reagent, TakaraBio) with two plasmids (2.25 μg each) expressing complementarily tagged (SYFP21-215 and SYFP2210-238) enzymes (whose interactions we wish to assess), along with a plasmid (0.5 μg) expressing LSSmOrange (serving as a transfection marker). Twenty-four hours posttransfection, cells are imaged (at 60X) under an epifluorescence microscope (Nikon TE Eclipse 2000) equipped with a stage-top incubation system (Tokai HIT) that maintains the cells at 37 °C in a 5% CO2 atmosphere. Cells are imaged using a 60/1.2 NA oil immersion objective in both LSSmOrange and YFP channels. A preliminary assay identified combinations of enzyme pairs/labeling termini pairings that gave clear BiFC signals; these are then subjected to detailed quantitative analysis.
3
Quantitative Imaging of Membrane Contacts
4
Live-cell Imaging of Molecular Co-recruitment
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Co-recruitment assays were performed using Eclipse Ti microscope (Nikon, Japan) with a 100 X TIRF objective (1.0 X zoom and 4X4 binning) in TIRF mode and PCO-Edge 4.2 BI sCMOS camera (PCO, Germany), driven by NIS Elements software (Nikon) and equipped with 440 nm, 514 nm, and 561 nm laser lines. Time lapse imaging was performed at 2 min intervals for 20 min, with 100 nM rapamycin addition after the fifth time point. All live cell imaging was conducted at 37 °C, 5% CO2 and 90% humidity with a stage top incubation system (Tokai Hit). Vitamin and phenol red-free media (US Biological) supplemented with 2% FBS were used in imaging to reduce background and photobleaching. Adequate co-expression of all relevant plasmids was confirmed by fluorescent imaging at appropriate wavelengths. To minimize variability due to relative expression levels, only cells showing at least 30% increase in mCherry intensity after addition of rapamycin were considered for quantification. All image processing and analyses for co-recruitment assays were performed using Metamorph (Molecular Devices, Sunnyvale, CA, USA) and FIJI software (NIH, Bethesda, MD, USA). Co-recruitment assay graphs show means ±95% confidence interval with n≥40 different cells pooled from at least three independent experiments. For information on plasmids used in these assays, see the Key Resources Table.
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