Lentiviral Transduction of HUVECs for Live Imaging

Lentiviral particles were generated by transfecting HEK293T cells with the lentiviral expression construct with 3rd generation packaging plasmids using TransIT^®-LT1 transfection reagents (Mirus Bio LLC, Madison, WI). The supernatant containing the lentiviral particles was harvested 48-72 h post transfection. HUVECs were transduced with LifeAct-mTurquoise construct (as described in Goedhart et al. 2012 [11 (link)]) cloned into a Lentiviral vector (pLV) overnight, selected with puromycin and grown to confluence on fibronectin-coated Lab-Tek Chambered 1.0 borosilicate coverglass slides (ThermoFisher) in EGM™-2 medium (Lonza). Live cell imaging was done with an inverted NIKON Eclipse Ti equipped with a 60 × 1.49 NA Apo TIRF (oil) objective, perfect focus system, CFP and mCherry filter cubes and an Andor Zyla 4.2 plus sCMOS camera. An Okolab cage incubator and humidified CO₂ gas chamber set to 37 °C and 5% CO₂ were used during imaging. Frames were taken every minute for 4–8 h.

Free full text: Click here

van der Wijk A.E., Georgakopoulou T., Majolée J., van Bezu J.S., van der Stoel M.M., van het Hof B.J., de Vries H.E., Huveneers S., Hordijk P.L., Bakker E.N, & van Bavel E. (2020). Microembolus clearance through angiophagy is an auxiliary mechanism preserving tissue perfusion in the rat brain. Acta Neuropathologica Communications, 8, 195.

Publication 2020

TransIT®-LT1 transfection reagents EGM™-2 medium Eclipse Ti cage incubator

Apo a 1 Cell Cubes Fibronectin 1 Frames Plasmids Puromycin Transfection Vector

Corresponding Organization : Academic Medical Center

Other organizations : University of Amsterdam, Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, Amsterdam Neuroscience

Top 5 similar protocols

Variable analysis

independent variables

Lentiviral expression construct with 3rd generation packaging plasmids
LifeAct-mTurquoise construct cloned into a Lentiviral vector (pLV)

dependent variables

Lentiviral particle production
Transduction of HUVECs with LifeAct-mTurquoise construct
Live cell imaging of HUVECs expressing LifeAct-mTurquoise

control variables

Transfection of HEK293T cells using TransIT®-LT1 transfection reagents
Overnight transduction of HUVECs with LifeAct-mTurquoise construct
Selection of transduced HUVECs with puromycin
Growth of HUVECs to confluence on fibronectin-coated Lab-Tek Chambered 1.0 borosilicate coverglass slides in EGM™-2 medium
Live cell imaging conditions (inverted NIKON Eclipse Ti microscope, 60 × 1.49 NA Apo TIRF (oil) objective, CFP and mCherry filter cubes, Andor Zyla 4.2 plus sCMOS camera, Okolab cage incubator and humidified CO2 gas chamber set to 37 °C and 5% CO2)

controls

No positive or negative controls were explicitly mentioned in the protocol.

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

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?

Revolutionizing how scientists
search and build protocols!