Live cell imaging of H2b-mcherry expressing MEFs was performed on glass-bottom 6-well plates (MatTek, Ashland, MA) using a Nikon Ti-E inverted microscope attached to a CoolSNAP CCD camera (Photometrics). Fluorescence and differential interference contrast (DIC) images were acquired every 7 minutes, and images were analyzed using NIS elements software (Nikon) and ImageJ software (NIH). For confocal imaging, MEFs were grown on 35mm glass bottom plates (MatTek, Ashland, MA) and DIC images were acquired every 5 minutes with the Ultraview Vox spinning disc confocal system (Perkin Elmer) equipped with a Yokogawa CSU-X1 spinning disc head, and EMCCD camera (Hamamatsu C9100-13), and coupled with a Nikon Ti-E microscope. Image analysis was performed with Volocity software (Perkin Elemer). All imaging was carried out in incubation chambers at 5% CO2 and 37°C.
C9100 13
Manufactured by Nikon
The C9100-13 is a high-performance charge-coupled device (CCD) camera designed for scientific and industrial applications. It features a back-illuminated sensor with a large field of view and high quantum efficiency, making it suitable for a variety of imaging tasks. The camera provides a resolution of 1344 x 1024 pixels and can capture images at a frame rate of up to 10 frames per second. The C9100-13 is equipped with a range of connectivity options, including FireWire and USB interfaces, and supports various image acquisition and processing software.
Automatically generated - may contain errors
Lab products found in correlation
Ultraview vox spinning disk confocal system, by PerkinElmer (7 mentions)
Volocity software, by PerkinElmer (7 mentions)
Csu x1 spinning disk head, by Hamamatsu Photonics (5 mentions)
Ti e microscope, by Nikon (5 mentions)
Alexa fluor 568 goat anti mouse secondary, by Thermo Fisher Scientific (4 mentions)
Alexa fluor 488 goat anti rabbit secondary, by Thermo Fisher Scientific (4 mentions)
P35g 1.5 20 c, by MatTek (4 mentions)
D1306, by Thermo Fisher Scientific (3 mentions)
Csu x1 spinning disc head, by Hamamatsu Photonics (2 mentions)
Anti lamp1, by BD (2 mentions)
C2206, by Merck Group (2 mentions)
A11034, by Thermo Fisher Scientific (2 mentions)
Ti e microscope, by PerkinElmer (2 mentions)
A11031, by Thermo Fisher Scientific (2 mentions)
35 mm glass bottom plate, by MatTek (1 mentions)
Ultraview vox spinning disc confocal system, by PerkinElmer (1 mentions)
Glass bottom 6 well plate, by MatTek (1 mentions)
Nis elements software, by Nikon (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
P06g 1.5 20 f, by MatTek (1 mentions)
9 protocols using c9100 13
1
Live-cell Imaging of H2b-mCherry MEFs
2
Live-cell Imaging of Lysosomal Dynamics
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Cells were plated on glass-bottom dishes (P06G-1.5-20-F, MatTek) in full medium overnight and were then imaged by time-lapse microscopy at 37 °C and 5% CO2 in a live-cell incubation chamber. LLOMe (300 nM) was added to the medium, and cells were imaged for 1 h at 15-min time intervals, as indicated. For LysoTracker staining experiments, MEFs were incubated with 100 nM LysoTracker Red DND-99 (L7528, Thermo Fisher Scientific, Waltham, MA) for 10 min followed by three PBS washes prior to treatment with or without 100 nM concanamycin A. Cells were then imaged by confocal microscopy. Fluorescence confocal micrographs were acquired using the Ultraview Vox spinning-disk confocal system (PerkinElmer, Waltham, MA) equipped with a Yokogawa CSU-X1 spinning-disk head and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13) coupled to a Nikon Ti-E microscope equipped with a CFI Plan Apo VC ×60 oil objective. Z stacks (0.5-μm steps) were acquired with a Piezo z stack drive controlled by a nano drive (Mad City Lab, Madison, WI).
3
Immunofluorescence Imaging of Cell Adhesion Proteins
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The following antibodies were used for immunofluorescence (IF): anti-E-cadherin (1:100; Cell Signaling), anti-β-catenin (1:100; Sigma-Aldrich), anti-Lamp1 (1:100; BD Biosciences), Alexa Fluor 568 goat anti-mouse secondary (1:500; Life Technologies), and Alexa Fluor 488 goat anti-rabbit secondary (1:500; Life Technologies). IF was performed as described previously (Overholtzer et al., 2007 (link)). Briefly, cells were cultured on glass-bottom dishes (P35G-1.5-20-C; MatTek) and were fixed in 1:1 methanol/acetone for 5 min at −20°C, followed by three 5-min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 hr, followed by incubation with primary antibodies at 4°C overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (1:1,000; Life Technologies). Confocal microscopy was performed with the Ultraview Vox spinning-disk confocal system (PerkinElmer) equipped with a Yokogawa CSU-X1 spinning-disk head and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13) coupled to a Nikon Ti-E microscope; image analysis was performed using Volocity software (PerkinElmer).
4
Lipid Peroxidation Imaging in Cells
5
Immunofluorescence Microscopy of Cellular Targets
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Immunofluorescence was performed on cells cultured on glass‐bottom dishes (P35G‐1.5‐20‐C, MatTek, Ashland, MA, USA), as described previously.7 Briefly, cells were fixed in 1:1 methanol/acetone for 5 min at −20℃, followed by three 5‐min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 h, followed by incubation with primary antibodies at 4℃ overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (D1306, Life Technologies, Carlsbad, CA, USA). Confocal microscopy was performed with the Ultraview Vox spinning‐disk confocal system (PerkinElmer) equipped with a Yokogawa CSU‐X1 spinning disk head and an electron‐multiplying charge‐coupled device camera (Hamamatsu C9100‐13) coupled to a Nikon Ti‐E microsope; image analysis was done using Volocity software (PerkinElmer). The following antibodies were used for immunofluorescence: anti‐phospho‐mTOR (Ser2448) (5536, Cell Signaling, Beverly, MA, USA), anti‐LAMP1 (555798, BD Biosciences, San Jose, CA, USA), anti‐BrdU (5292, Cell Signaling, Beverly, MA, USA), Alexa Fluor 568 goat anti‐mouse secondary (A‐11031, Life Technologies, Carlsbad, CA, USA), and Alexa Fluor 488 goat anti‐rabbit secondary (A‐11034, Life Technologies, Carlsbad, CA, USA).
6
Immunofluorescence Imaging of β-Catenin and Lamp1
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The following antibodies were used for immunofluorescence (IF): anti-β-catenin (1:100; C2206; Sigma-Aldrich), anti-Lamp1 (1:100; 555798; BD Biosciences), Alexa Fluor 568 goat anti-mouse secondary (1:500; A-11031; Life Technologies), and Alexa Fluor 488 goat anti-rabbit secondary (1:500; A-11034; Life Technologies). IF was performed on cells cultured on glass-bottom dishes (P35G-1.5-20-C; MatTek), as described previously (Overholtzer et al., 2007 (link)). Briefly, cells were fixed in 1:1 methanol/acetone for 5 min at −20°C, followed by three 5-min PBS washes and blocking in 5% BSA, 100 mM glycine in PBS for 1 hr, followed by incubation with primary antibodies at 4°C overnight. Samples were then incubated with secondary antibodies and counterstained with DAPI (1:1,000; D1306; Life Technologies). Confocal microscopy was performed with the Ultraview Vox spinning-disk confocal system (PerkinElmer) equipped with a Yokogawa CSU-X1 spinning-disk head and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13) coupled to a Nikon Ti-E microscope; image analysis was done using Volocity software (PerkinElmer).
7
Immunofluorescence Imaging of β-Catenin and Lamp1
8
Lipid Peroxidation Imaging in Cells
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Cells were plated on glass-bottom dishes and treated the next day. For C11-BODIPY581/591 imaging, cells were washed twice with Hank’s Balanced Salt Solution (HBSS) (14025-092; ThermoFisher) 24 hours after treatment, stained in 5μM C11-BODIPY581/591 in HBSS for 10 minutes at 37°C and 5% CO2, and again washed twice in HBSS. Cells were imaged at 37°C and 5% CO2 using the Ultraview Vox spinning-disk confocal system (PerkinElmer) equipped with 488nm and 568nm lasers and an electron-multiplying charge-coupled device camera (Hamamatsu C9100-13), and attached to a Nikon Ti-E microscope. For cPLA2 imaging shown in Figure 2f , a single confocal plane is shown from the indicated time points. For C11-BODIPY581/591 imaging in Extended Data Figure 1 , maximum projections are shown. Images were acquired and processed using Volocity software (Perkin Elmer, version 5.2.0).
9
Live-Cell Confocal Imaging of Lysosomal Dynamics
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