Nuclear F-actin induction was observed in live cells by infecting HFFs stably expressing LifeAct-GFP-NLS with WT HCMV (MOI of 3). Directly following infection, cells were imaged at the Nikon Imaging Center at Harvard Medical School using a Nikon Ti epifluorescence microscope equipped with an Orca-ER cooled CCD camera (Hamamatsu) and an incubation chamber (OkoLab). Images were taken every 3 min up to 28 hpi.
Incubation chamber
Manufactured by Okolab
Sourced in Italy
The Incubation chamber is a laboratory equipment designed to provide a controlled environment for the incubation of biological samples. Its core function is to maintain a stable temperature, humidity, and atmospheric conditions within the enclosed space, as required for the specific experimental or culturing needs.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (2 mentions)
Orca flash 4, by Hamamatsu Photonics (2 mentions)
Orca er cooled ccd camera, by Hamamatsu Photonics (1 mentions)
Glass bottomed dish, by MatTek (1 mentions)
Eclipse ti spinning disk, by Nikon (1 mentions)
Celltracker red cmptx, by Thermo Fisher Scientific (1 mentions)
Eclipse te2000 e, by Nikon (1 mentions)
Metamorph software, by Universal Imaging (1 mentions)
Nis elements version 4, by Nikon (1 mentions)
Eclipse ti e inverted microscope, by Nikon (1 mentions)
Nis elements br 3, by Nikon (1 mentions)
Orca flash4.0 v2, by Hamamatsu Photonics (1 mentions)
Eclipse ti e, by Nikon (1 mentions)
50 objective, by Zeiss (1 mentions)
Csu x filter wheel, by Yokogawa (1 mentions)
Hcs lipidtox deep red, by Thermo Fisher Scientific (1 mentions)
Ethidium homodimer, by Thermo Fisher Scientific (1 mentions)
Fluorobrite dmem, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fugene 6, by Promega (1 mentions)
12 protocols using incubation chamber
1
Live Imaging of Nuclear F-actin Dynamics
2
Live Embryo Imaging Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Injected embryos were transferred to the environmental chamber of the spinning disk microscope (Andor Revolution XD System with a Nikon Eclipse Ti Spinning Disk) and imaged for 2 days. Twenty-one z-stacks per time step (20 or 30 min) were taken, with two channels (567 nm excitation for ESC visualisation, and bright field). Temperature (37°C), CO2 concentration (7%) and fluorescence exposure (148 ms of 567, 300 ms bright field) were standardised. Prior to each imaging experiment, the incubation chamber (Oko Lab) was allowed to stabilise to 37°C. The CO2 concentration was generated by an active mixer (Life Imaging Sciences) and humidified before supply to the sample. Embryos were immobilised using a 118×118 µm polyester mesh (Plastok Group) in a glass-bottomed dish (MatTek Corporation). An Andor 85 camera recorded images with magnification through a Plan Fluor 40×/1.3 NA oil lens. Each experiment was set up using Andor IQ Software. A multi-position map was created: every embryo was manually assigned an x-y-z location at its centre and visited (starting from the upper-most plane) by the 40× lens at each time point of data acquisition. Channels were sequentially acquired per z-section. Each image collected data in 502×501 (width×height) pixels, 2 μm per pixel.
3
Live Cell Imaging of Cellular Topographies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For live cell imaging, cells adhering onto the topographies where loaded with the dye CellTracker Red CMPTX (ThermoFischer) and placed in an incubation chamber (Okolab) at 37 °C, 5%CO2 and H2O saturated. Z-stacks were acquired at regular time intervals with a 20 × magnification objective. Particle tracking was done on vertical maximum projection of the z-stacks using Imaris software (Bitplane).
4
Time-lapse Imaging of Cell Migration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at low density in a 6-well plate (2x104 cells/well). Time-lapse imaging of cell migration was performed on a NIKON Eclipse TE2000-E inverted microscope equipped with an incubation chamber (OKOLab) for temperature and CO2 control. Movies were acquired by a Cascade II 512 (Photometrics) CCD camera controlled by Metamorph Software (Universal Imaging Corporation) using a 20x magnification objective. Tracking of cells was performed using the “Manual Tracking” plug-in distributed with ImageJ software. Data were analyzed using one-way ANOVA followed by Bonferroni's Multiple Comparison Test performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego California USA).
5
Exosome-induced Tube Formation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Analysis of capillary-like tube formation was performed using semisolid medium Cultrex® Basement Membrane Extract (BME, Trevigen, Gaithersburg, MD, USA). hFM-MSCs were detached, counted, and seeded at a density of 30,000 cells/cm2 in a BME-coated 96-well plate. Cells were maintained in basal medium (DMEM, 2 mM Ultraglutamine, 1% penicillin-streptomycin solution) supplemented with exosomes or vehicle (DPBS) as negative control. The plate was introduced into the incubation chamber (OKOlab, Naples, Italy) of the microscope station to maintain CO2, temperature, and humidity in controlled conditions, and time-lapse acquisitions were performed over 24 h with a Nikon Inverted Microscope Eclipse Ti-E (Nikon Instruments, Tokyo, Japan) equipped with a Digital Sight camera DS-03 linked to a computer driving the imaging software NIS-Elements version 4.30 (Nikon Instruments). Total well areas were captured, and tube length was measured using NIS-Elements software tools. At least 3 independent experiments were performed for each point.
6
Cell Migration Tracking Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Migration assays were conducted on an Eclipse Ti-E inverted full-motorized microscope (Nikon) equipped with an incubation chamber (OKOlab, Pozzuoli, Italy) maintained at 37 °C with 5% CO2. Movies were acquired by an ORCA Flash 4.0 V2 digital CMOS camera (EPI light path, Hamamatsu Photonics, Japan) controlled by NIS-Elements BR 3.0 software (Nikon). Cell migration was recorded for 24 h. Single cells’ tracking was conducted using the “Manual Tracking” plugin of ImageJ software (NIH, Bethesda, MA, USA).
7
Raman Spectroscopy of Organoids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spectral Raman mapping was performed on a customized inverted WITec alpha300 R Raman system (WITec GmbH, Ulm, Germany) equipped with a green laser (532 nm) and a CCD spectrograph with a grating of 600 g/mm. An incubation chamber (Okolab S.R.L.) was integrated into the setup to keep the organoids constantly at 37 °C. Images were acquired from at least three organoids at a laser power of 58 mW, an integration time per spectrum of 0.2 s and a pixel resolution of 1 × 1 μm at a size of 50 × 50 µm. All measurements were acquired with a 50× objective (Carl Zeiss AG, Oberkochen, Germany). Reference spectra of cytochrome c (derived from bovine heart, Sigma Aldrich), cis and vtx were recorded as single spectra with an accumulation of 10 spectra and 0.5 s integration time.
8
Visualizing Centrosome Calcium Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells stably expressing actin-GCaMP6s were plated on 35-mm glass-bottom dishes and synchronized using thymidine-nocodazole protocol. Before the second release, cells were loaded with 2 μM diazo-2-acetoxymethyl ester (diazo-2-AM; a kind gift from Alexei Tepikin, University of Liverpool) for at least 30 min at 37°C.
Cells were then released and examined on a 3i Marianas spinning-disk confocal microscope equipped with a Zeiss AxioObserver Z1, a ×40/1.3 Plan-Apochromat oil immersion objective, and a 3i Laserstack as excitation light source (405 nm for diazo-2-AM photolysis; 488 nm for actin-GCaMP6s). Emitted light was collected through single bandpass filters (CSU-X filter wheel; Yokogawa) onto a CMOS camera (Orca Flash 4.0; Hamamatsu). Experiments were carried out at 37°C and 5% (v/v) CO2 (incubation chamber from Okolab, Naples, Italy).
Photolysis of diazo-2-AM was performed by illumination with 405 nm laser light at 10% power for 10 ms for rapid chelation of Ca2+ at the centrosome or cytoplasm (elliptical region of interest) after 1–3 frames. Then, cell division was monitored by collecting images every minute using SlideBook v.6 software and processed on ImageJ and GraphPad Prism 6.
Cells were then released and examined on a 3i Marianas spinning-disk confocal microscope equipped with a Zeiss AxioObserver Z1, a ×40/1.3 Plan-Apochromat oil immersion objective, and a 3i Laserstack as excitation light source (405 nm for diazo-2-AM photolysis; 488 nm for actin-GCaMP6s). Emitted light was collected through single bandpass filters (CSU-X filter wheel; Yokogawa) onto a CMOS camera (Orca Flash 4.0; Hamamatsu). Experiments were carried out at 37°C and 5% (v/v) CO2 (incubation chamber from Okolab, Naples, Italy).
Photolysis of diazo-2-AM was performed by illumination with 405 nm laser light at 10% power for 10 ms for rapid chelation of Ca2+ at the centrosome or cytoplasm (elliptical region of interest) after 1–3 frames. Then, cell division was monitored by collecting images every minute using SlideBook v.6 software and processed on ImageJ and GraphPad Prism 6.
9
Placental Explant Lipid Droplet Imaging
10
Live-Cell Imaging of Transfected HeLa Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells were seeded onto 12-well #1.5 glass-bottom plates (Cellvis) and transiently transfected with FuGENE 6 (Promega), following the manufacturer’s instructions. Before imaging, cells were exchanged into imaging media: FluoroBrite DMEM (Thermo Fisher Scientific) supplemented with 10% FBS and 4 mM GlutaMAX (Thermo Fisher Scientific). Images were acquired as a series of 0.6-μm z-stacks with a Plan Apo 40×/0.95 Corr [Differential Interference Contrast (DIC) N2/40× I] 227.5 nm per pixel objective (Nikon) on a Nikon Ti-E inverted CSU-22 spinning disk confocal microscope equipped with an incubation chamber (Okolab), providing a humidified atmosphere at 37°C with 5% CO2. Images were processed using Fiji (64 (link)).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!