Journal: eLife

Article Title: Quantitative live-cell imaging and computational modeling shed new light on endogenous WNT/CTNNB1 signaling dynamics

doi: 10.7554/eLife.66440

Figure Lengend Snippet:

Article Snippet: The following plasmids are available from Addgene: pX459-CTNNB1-ATG (#153429), pX459-CTNNB1-S45 (#164587), pRepair-SGFP2-CTNNB1 (#153430), pRepair-mScI-CTNNB1 (#153431), pRepair-SYFP2-CTNNB1 (#153432), pRepair-mTq2-CTNNB1 (#153433).

Techniques: Transfection, Construct, Recombinant, Staining, Software, Modification

Journal: Analytical and bioanalytical chemistry

Article Title: Optical approaches for single cell and subcellular analysis of GPCR-G protein signaling

doi: 10.1007/s00216-019-01774-6

Figure Lengend Snippet: FRET and BRET based biosensors

Article Snippet: Therefore, appropriate FRET or BRET probes can be designed to examine intermolecular receptor-G proteins and G protein-G protein interactions, thus measuring GPCR and G protein activation in real-time [ 126 , 127 , 122 ]. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Biosensor Name Target Number of molecules Physical type Pubmed ID Addgene plasmid # Gαi3 v1 Gαi Bimolecular or other FRET 16371464 Gαi-Gβ1 Gαi Bimolecular or other FRET 14673086 Gαi-Gγ2 Gαi Unimolecular FRET 14673086 Gαi2 Sensor v2 Gαi Bimolecular or other FRET 26799488 #69624 Gαi1 Sensor v2 Gαi Bimolecular or other FRET 26799488 #69623 RLucll-117-Gαs + GFP10-GY1 Gαs Bimolecular or other BRET 27499021 Gαs Sensor Gαs Bimolecular or other FRET 16963443 Gαq Sensor (v2) Gαq Bimolecular or other FRET 21619590 Gα13 Sensor (v2) Gα13 Unimolecular FRET 29505611 #112933 HCN2-camps cAMP Unimolecular FRET 17038640 Epac1-camps cAMP Unimolecular FRET 15231839 Epac2-camps cAMP Unimolecular FRET 15231839 mICNBD-FRET cAMP Unimolecular FRET 27003291 CAMYEL cAMP Unimolecular BRET 17283075 BFP-PMCA-GFP Ca 2+ Unimolecular FRET 17901055 AKAR4 PKA Unimolecular FRET #61619 CKAR PKC Unimolecular FRET 12782683 #14860 Open in a separate window FRET and BRET based biosensors 5.1.

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Live Cell Multicolour Lifetime Imaging Using Genetically Encodable Fluorophores

doi: 10.1101/2022.10.06.511114

Figure Lengend Snippet: (a) Live cells (middle panels) expressing a) tandem mTFP1-linker-mTurquoise2 (mTQ2), b) bi-cistronic mTFP1-T2A-mTQ2 and c) co-expression of mTFP1 and mTQ2 were tested to unmix both populations of plasmids. Scale bar 5 μm. Bar charts (right panels) plotting the lifetimes of mTFP1 (dark blue, 2.8 ns) and mTQ2 (cyan, 4.12 ns) when expressed alone were employed as a reference as explained in methods. The percentage obtained for the three cases depicted were 52+/−3%, n = 10; 49+/−4, n = 10 and 50+/−4%, n = 10; respectively. (b) A biological example shows live cells mimicking the HIV-1 virological synapse co-expressing CD4-mTFP1 and CXCR4-mTQ2 (target cells) and HXB2 Env and Gag-mCherry (effector cells). Intensity (left panel) and FLIM images (middle and right micrographs) are depicted showing that one can separate spectrally similar fluorescent proteins. Scale bar 5 μm. Bar diagrams are shown with quantification of the proportion of components colocalizing without Gag-mCherry (bottom left panels) and with Gag-mCherry (bottom right panels). Proportions of CD4-mTFP1 (60+/−10%, n = 5) and coreceptor CXCR4-mTQ2 (40+/−10%, n = 5) differ as compared to other regions where these receptors diffuse freely.

Article Snippet: Most of the DNA plasmids used in this article were obtained via Addgene ( https://www.addgene.org/ ; see and ). hCXCR4 and hCCR5 were cloned into pmTurquoise2-C1 (#60560) vector by ligating NheI/AgeI fragments into the corresponding sites of the vector, to make hCXCR4-mTQ2 and hCCR5-mTQ2.

Techniques: Expressing

Journal: bioRxiv

Article Title: Live Cell Multicolour Lifetime Imaging Using Genetically Encodable Fluorophores

doi: 10.1101/2022.10.06.511114

Figure Lengend Snippet: Cartoon showing cells co-cultured together co-expressing nine different FP plasmids (three by each cell). The cells were mixed after co-transfection of three different plasmids in each of the three initial wells. (b) The micrographs of live cells expressing nine different plasmids in three different spectral channels are shown. In the blue channel one has cells expressing histone H4 H4-mTFP1 and histone H2B-mTQ2. In the green channel there are cells expressing LaminB-mWasabi, cytosolic mAmetrine and EMTB-EGFP. In the red channel one has cells expressing cytosolic LSSmOrange, Mito-LSSmKate2, Alphactinin-mCherry and Cytosolic mKate2. Scale bar 10 μm. (c) the Intensity micrographs (left columns) together with the overall lifetime images (middle panels) and lifetime unmixed panels (right panels) are depicted. The corresponding pixel lifetime histograms before and after unmixing are also shown. The nine different fluorescent species were separated successfully.

Article Snippet: Most of the DNA plasmids used in this article were obtained via Addgene ( https://www.addgene.org/ ; see and ). hCXCR4 and hCCR5 were cloned into pmTurquoise2-C1 (#60560) vector by ligating NheI/AgeI fragments into the corresponding sites of the vector, to make hCXCR4-mTQ2 and hCCR5-mTQ2.

Techniques: Cell Culture, Expressing, Cotransfection