U2OS or HEK293 cells expressing GFP–FAM35A or GFP–C20ORF196 were cultured at 37 °C in CO2-independent medium (Invitrogen) containing 10% FBS in a temperature-controlled container in glass-bottom dishes (MatTek). Laser microirradiation was carried out with the Micro-Point Laser Illumination and Ablation System (ANDOR) coupled to a Leica DMI8 microscope with a 63 × CS2 oil immersion objective. Images were acquired with ANDOR IQ3 software through an ANDOR IXON camera with ANDOR Dragonfly system.
Dragonfly system
Manufactured by Oxford Instruments
The Dragonfly system is a versatile laboratory instrument designed for high-precision material analysis. It provides advanced characterization capabilities through its integrated suite of analytical tools. The Dragonfly system is suitable for a wide range of applications, offering reliable and accurate data acquisition.
Automatically generated - may contain errors
Lab products found in correlation
Live cell imaging solution, by Thermo Fisher Scientific (3 mentions)
Co2 independent medium, by Thermo Fisher Scientific (1 mentions)
Micropoint laser illumination and ablation system, by Oxford Instruments (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
Glass bottom dishes, by MatTek (1 mentions)
Scmos camera, by Oxford Instruments (1 mentions)
Thapsigargin, by Thermo Fisher Scientific (1 mentions)
Bapta am, by Thermo Fisher Scientific (1 mentions)
Lsm 880 airyscan, by Zeiss (1 mentions)
Eclipse ti2, by Oxford Instruments (1 mentions)
7 protocols using dragonfly system
1
GFP-tagged Protein Microirradiation Imaging
2
Quantifying Fluorescent Nuclear Protein Arrays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U2OS-265 cells expressing mCherry-LacR or GFP-rTetR were cultured at 37 °C in DMEM medium containing 10% FBS. Images were acquired with Imaris software (Bitplane) through an ANDOR IXON camera with an ANDOR Dragonfly system. The area of the array was analyzed using Imaris (Bitplane). Cells were chosen at random for the analysis of the intensity of the indicated signals. The region of the array was defined by the mCherry-LacR signal and measured using Imaris software. The accumulated signals were measured by determining the mean fluorescent intensity of the array region and normalizing this value to the mean fluorescent intensity of the entire nucleus. For each cell, a separate region was measured for background subtraction. The accumulated signal was calculated by the following formula: I = (Iarray – Inu)/(Inu – Ib), where Iarray is the mean fluorescence intensity of the array region, Ib is the mean fluorescence intensity of the background, and Inu is the mean fluorescence intensity of the nucleus. All intensity analysis was performed on unprocessed images using ImageJ software.
3
TIRF Imaging of Arrestin Translocation
4
Live-cell Imaging of VPS13A Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Just before imaging, the growth medium was removed and replaced with prewarmed Live Cell Imaging solution (Life Technologies). Imaging was carried out at 37 °C and 5% CO2. Spinning-disk confocal microscopy was performed using an Andor Dragonfly system equipped with a PlanApo objective (63×, 1.4 numerical aperture, oil) and a Zyla sCMOS camera. For obtaining images of COS7 cells in suspension, COS7 were trypsinized and replated in MatTeks 10 min before imaging. Images were analyzed in FIJI. A Gaussian Blur filter was applied to some of the images presented. The tabular data for the graphs relative to the localization of VPS13A to either mitochondria or the PM are posted in a public database at https://doi.org/10.5281/zenodo.6568287 .
5
Live-Cell Imaging and Cytosolic Calcium Monitoring
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Just before imaging, the growth medium was removed and replaced with live-cell imaging solution (Life Technologies). All live-cell imaging was performed at 37°C and 5% CO2. Spinning-disk confocal microscopy was performed using an Andor Dragonfly system equipped with a plan apochromat objective (63×, 1.4 NA, oil) and a Zyla scientific CMOS camera. For the hypotonic lysis experiments, live-cell imaging solution was replaced with distilled water, and cells were imaged at a rate of 0.5 Hz.
For experiments involving cytosolic Ca2+ changes, COS7 cells were seeded and transfected as explained above. To acutely increase cytosolic Ca2+, 2 µM thapsigargin (Life Technologies) was added to the live-cell imaging solution (Life Technologies), which contains 1.8 mM Ca2+, and cytosolic Ca2+ was monitored by the intensity of the RFP genetically encoded Ca2+ indicator for optical imaging (plasmid was a gift from R. Campbell, University of Alberta, Edmonton, AB, Canada; Addgene catalog no. 45494). To decrease cytosolic Ca2+, 4 mM EGTA and 10 µM BAPTA-AM (Thermo Fisher Scientific) were added to the medium during imaging. Calcium experiments were repeated twice in at least four cells for each case. The general protocol for the imaging experiments described was deposited in protocols.io:https://dx.doi.org/10.17504/protocols.io.bvgmn3u6 .
For experiments involving cytosolic Ca2+ changes, COS7 cells were seeded and transfected as explained above. To acutely increase cytosolic Ca2+, 2 µM thapsigargin (Life Technologies) was added to the live-cell imaging solution (Life Technologies), which contains 1.8 mM Ca2+, and cytosolic Ca2+ was monitored by the intensity of the RFP genetically encoded Ca2+ indicator for optical imaging (plasmid was a gift from R. Campbell, University of Alberta, Edmonton, AB, Canada; Addgene catalog no. 45494). To decrease cytosolic Ca2+, 4 mM EGTA and 10 µM BAPTA-AM (Thermo Fisher Scientific) were added to the medium during imaging. Calcium experiments were repeated twice in at least four cells for each case. The general protocol for the imaging experiments described was deposited in protocols.io:
6
Live-cell Confocal Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Just before imaging, the growth medium was removed and replaced with prewarmed Live Cell Imaging solution (Life Technologies). Imaging was carried out at 37 °C and 5% CO2. Spinning-disk confocal microscopy was performed using an Andor Dragonfly system equipped with a PlanApo objective (63×, 1.4 numerical aperture, oil) and a Zyla sCMOS camera, airyscan imaging is performed using Zeiss LSM 880 Airyscan. Images were analyzed in FIJI.
7
Laser-Induced DNA Damage Tracking
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells or U2OS cells were seeded in 35 mm confocal dishes for 24 h and transfected with GFP-tagged proteins. After 24 h of transfection, the cells were irradiated with a 405 nm laser (Nikon Eclipse Ti2 and Andor Dragonfly system), and images were recorded for the indicated times. The fluorescence intensity of the laser tracks and the DNA fiber tract lengths were measured using ImageJ.
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!