Electroformation of Fluorescent GUVs

GUVs were prepared via electroformation as essentially described^{28 (link),29 (link)}. Lipid-coated ITO slides were dried under vacuum overnight. The following lipid mixtures (in mol%) were spread on two ITO slides [the fluorescent lipid Texas Red-DHPE (Life Technologies) was added for imaging purposes]: DOPC/Texas Red-DHPE: 99.75/0.25, DOPC/TR-DHPE/cholesterol: 74.75/0.25/25, 94.75/0.25/5 DOPC/TR-DHPE/DOPS, 74.75/0.25/5/20 DOPC/TR-DHPE/DOPS/cholesterol, 59.75/0.25/20/20, DOPC/TR-DHPE/DOPS/cholesterol, 94.75/0.25/5 DOPC/TR-DHPE/DOPA, 74.75/0.25/5/20 DOPC/TR-DHPE/DOPA/cholesterol, DOPC/TR-DHPE/DOPS/cholesterol, 94.75/0.25/5 DOPC/TR-DHPE/PI(4,5)P₂, 74.75/0.25/5/20 DOPC/TR-DHPE/PI(4,5)P₂/cholesterol, 69.75/0.25/10/20 DOPC/TR-DHPE/PI(4,5)P₂/cholesterol or 69.75/0.25/10/20 DOPC/TR-DHPE/PI(3)P/cholesterol. The two ITO slides were combined with the space between filled with sucrose (10%, w/v) and incubated at RT for 1.5 h (electroformation). The GUVs were added at room temperature to DyLight488-labeled rCPn0473 (100 µg/ml) and immediately observed on a confocal fluorescence microscope (Nikon Eclipse Ti-E with A1R confocal laser scanner, 60x oil objective, NA = 1.49). Image acquisition and analysis was performed with NIS-Elements (Nikon).

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Galle J.N., Fechtner T., Eierhoff T., Römer W, & Hegemann J.H. (2019). A Chlamydia pneumoniae adhesin induces phosphatidylserine exposure on host cells. Nature Communications, 10, 4644.

Publication 2019

Texas Red-DHPE TR-DHPE Eclipse Ti-E A1R confocal laser scanner NIS-Elements

Cholesterol Dhpe Dopa Dopc Dops Fluorescence microscope Lipid Sucrose Tr dhpe Vacuum

Corresponding Organization :

Other organizations : Heinrich Heine University Düsseldorf, University of Freiburg

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Variable analysis

independent variables

Lipid mixtures (in mol%): DOPC/Texas Red-DHPE: 99.75/0.25, DOPC/TR-DHPE/cholesterol: 74.75/0.25/25, 94.75/0.25/5 DOPC/TR-DHPE/DOPS, 74.75/0.25/5/20 DOPC/TR-DHPE/DOPS/cholesterol, 59.75/0.25/20/20, DOPC/TR-DHPE/DOPS/cholesterol, 94.75/0.25/5 DOPC/TR-DHPE/DOPA, 74.75/0.25/5/20 DOPC/TR-DHPE/DOPA/cholesterol, DOPC/TR-DHPE/DOPS/cholesterol, 94.75/0.25/5 DOPC/TR-DHPE/PI(4,5)P2, 74.75/0.25/5/20 DOPC/TR-DHPE/PI(4,5)P2/cholesterol, 69.75/0.25/10/20 DOPC/TR-DHPE/PI(4,5)P2/cholesterol or 69.75/0.25/10/20 DOPC/TR-DHPE/PI(3)P/cholesterol

dependent variables

Binding of DyLight488-labeled rCPn0473 (100 µg/ml) to the GUVs

control variables

Sucrose (10%, w/v) in the space between the two ITO slides during electroformation
Incubation time of 1.5 h at room temperature during electroformation
Nikon Eclipse Ti-E with A1R confocal laser scanner, 60x oil objective, NA = 1.49 for image acquisition and analysis

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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