Cells were grown on chambered cover glasses to 70–80% confluency, at which time cells were stimulated with IL-1β (1 μg/ml) or TNFα (1 μg/ml). Live cell images were immediately captured every 10 seconds for 1 hour (IL-1β stimulation) or 30 min (TNFα stimulation) under an Andor spinning disk confocal microscope system equipped with a Nikon Ti motorized microscope (with 60× oil objective), a CSUX1 Spinning Disk Confocal head (Yokogawa), an Andor iXon EMCCD camera and a Neo sCMOS camera. Images were analyzed by ImageJ. To quantify the number of cellular condensates, backgrounds of images were first subtracted by using the module of subtract background on ImageJ, and then condensates (~ 0.3–1 μm in diameter) in cells were spotted and counted using the plugin of SpotCounter on ImageJ (the threshold of fluorescent intensity in detecting NEMO puncta was set above that of free NEMO). For tracking the fusion of two condensates as shown in Figure 2E –F , trajectories and positions of condensates were obtained using the plugin of TrackMate on ImageJ. Cells that contained NEMO puncta after IL-1β or TNFα treatment represented > 90% of stimulated cells.
Spinning disk confocal microscope system
Manufactured by Nikon
Sourced in Japan
The Spinning disk confocal microscope system is a laboratory equipment designed for high-speed, high-resolution imaging applications. This system utilizes a spinning disk with multiple apertures to provide optical sectioning, allowing for the capture of focused images at different depths within a sample. The system is capable of delivering rapid image acquisition and real-time visualization of dynamic processes, making it a valuable tool for various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ti motorized microscope, by Nikon (2 mentions)
Ixon emccd camera, by Oxford Instruments (2 mentions)
Neo scmos camera, by Oxford Instruments (2 mentions)
Csu x1 spinning disk confocal head, by Yokogawa (2 mentions)
Viewrna ish cell plus assay kit, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (2 mentions)
Eclipse ti inverted microscope, by Nikon (2 mentions)
Millicell ez slide 4 well glass chambers, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Te2000 u inverted microscope, by Nikon (1 mentions)
Alexa fluor 568 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
6 protocols using spinning disk confocal microscope system
1
Imaging NEMO Condensate Dynamics
2
Imaging NEMO Condensate Dynamics
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Cells were grown on chambered cover glasses to 70–80% confluency, at which time cells were stimulated with IL-1β (1 μg/ml) or TNFα (1 μg/ml). Live cell images were immediately captured every 10 seconds for 1 hour (IL-1β stimulation) or 30 min (TNFα stimulation) under an Andor spinning disk confocal microscope system equipped with a Nikon Ti motorized microscope (with 60× oil objective), a CSUX1 Spinning Disk Confocal head (Yokogawa), an Andor iXon EMCCD camera and a Neo sCMOS camera. Images were analyzed by ImageJ. To quantify the number of cellular condensates, backgrounds of images were first subtracted by using the module of subtract background on ImageJ, and then condensates (~ 0.3–1 μm in diameter) in cells were spotted and counted using the plugin of SpotCounter on ImageJ (the threshold of fluorescent intensity in detecting NEMO puncta was set above that of free NEMO). For tracking the fusion of two condensates as shown in Figure 2E –F , trajectories and positions of condensates were obtained using the plugin of TrackMate on ImageJ. Cells that contained NEMO puncta after IL-1β or TNFα treatment represented > 90% of stimulated cells.
3
Zebrafish Embryo Generation and Observation
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Embryos were generated by natural pair-wise mating when the fish were 3~12 months old. The filters were switched off and breeding boxes were placed into the tanks, followed by exposure to light. The fish was left undisturbed for 15~30 min. Breeding boxes were collected and the embryos were transferred into clean petri-dishes with a fine fishing net and the embryos were maintained in Milli-Q water. Healthy, transparent and regular embryos were picked out at the 1~4 cell stage and were distributed into a 24-well microplate with 6~8 embryos per well depending on the assay. The morphological changes of embryos were observed using an Olympus Spinning Disk Confocal Microscope System (Nikon, Japan).
4
Visualizing Protein Trafficking Dynamics
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Cells were stained in serum-free media with the following vital dyes: Bodipy TR C5-ceramide-BSA (5 mM, 30 min; 40°C; Molecular Probes; Invitrogen) in HEPES medium, to visualize the Golgi Complex or Lysotracker Red (50 nM; 30 min; Molecular Probes, Invitrogen), to visualize lysosomes. After washing with warm serum-free media, cells were incubated with Alexa Fluor 488 (Life Technologies, Grand Island, NY)-labeled A1AT (100 µg/ml; 30 min), which was then removed by a second wash with warm serum-free media, and then visualized by live microscopy every 3 minutes for 2 h. Images were acquired using a Perkin-Elmer spinning disk confocal microscope system mounted on a Nikon TE 2000 U inverted microscope, using Nikon ×100 NA 1.4 oil immersion plan apochromatic objective. The excitation wavelength was set at 56 nm or 488 nm, and the emission filters used were 600/45 or 525/50 for all experiments. The system is equipped with an Andor EM-CCD system (South Windsor, CT).
5
ADIPINT Expression Analysis by RNA-FISH
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RNA-FISH assays for ADIPINT using the ViewRNA® ISH Cell Plus Assay Kit (Affymetrix) according to the manufacturer’s instructions. Type 6 probes against ADIPINT were ordered from the same supplier. Briefly, hADSC were cultured in Millicell EZ SLIDE 4-well glass chambers (Merck Chemicals and Life Science AB). At day 13 of differentiation, the adipocytes were washed with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde for 20 min at room temperature. Subsequently, probes were hybridized for 2 h at 40 °C. After hybridization and amplification steps with pre-amplifier, amplifier, and linked labelled probe, the probes were detected using Alexa® Fluor 650 dyes, according to the manufacturer’s instructions. Cells were stained with 1:2500 BODIPY 493/503 and Hoechst 33342 for 20 min before mounting with ProLong Gold Antifade Mountant (ThermoFisher). Images were obtained with a Nikon spinning disk confocal microscope system (Nikon) equipped with a Nikon ECLIPSE Ti inverted microscope and a 1.4 NA x60 oil immersion objective (Nikon), under the control of NIS-Elements AR version 4 software.
6
Adipocyte Molecular Profiling via RNA-FISH and Immunofluorescence
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RNA-FISH assays for ADIPINT and immunofluorescence for PC was combined performed using the ViewRNA® ISH Cell Plus Assay Kit (Affymetrix) according to the manufacturer's instructions. Type 6 probes against ADIPINT were ordered from the same supplier. Briefly, hADSC were cultured in Millicell EZ SLIDE 4-well glass chambers (Merck Chemicals and Life Science AB). At day 13 of differentiation, the adipocytes were washed with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde for 20 min at room temperature. Following incubations with α-PC (PA5-50101) at 1:50 for 2 hours at room temperatures, the cells were incubated with Alexa Fluor® 568-conjugated secondary antibodies (A11011 , ThermoFisher) at 1:1000 for 1h at room temperature. Subsequently, probes were hybridized for 2 hours at 40°C. After hybridization and amplification steps with pre-amplifier, amplifier, and linked labelled probe, the probes were detected using Alexa® Fluor 650 dyes, according to the manufacturer's instructions. Cells were stained with 1:2500 BODIPY 493/503 and Hoechst 33342 for 20 minutes before mounting with ProLong Gold Antifade Mountant (ThermoFisher). Images were obtained with a Nikon spinning disk confocal microscope system (Nikon) equipped with a Nikon ECLIPSE Ti inverted microscope and a 1.4 NA 60x oil immersion objective (Nikon), under the control of NIS-Elements AR version 4 software.
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