Journal: Current protocols in neuroscience

Article Title: Protocol for assessing neuron-astrocyte spatial interactions using the neuron-astrocyte proximity assay

doi: 10.1002/cpns.91

Figure Lengend Snippet: Representative image settings used in the HEK293T cell experiments

Article Snippet: CMV mCherry GFP BGH , Octeau et al., 2018 , Addgene; plasmid #92280.

Techniques:

Journal: Current protocols in neuroscience

Article Title: Protocol for assessing neuron-astrocyte spatial interactions using the neuron-astrocyte proximity assay

doi: 10.1002/cpns.91

Figure Lengend Snippet: Bleed through and cross-talk (SBT) calculations to measure sensitized emission FRET (SE-FRET). A. Diagram of the two channels’ imaging conditions for the GFP alone, i.e. donor bleed through SBT model. B. Diagram of the two channels’ imaging conditions for the mCherry alone, i.e. acceptor cross-talk SBT model. C. Diagram of the three channels’ imaging conditions for the sensitized emission FRET experiment between GFP and mCherry. D. Förster equation used to calculate FRET efficiency, E.

Article Snippet: CMV mCherry GFP BGH , Octeau et al., 2018 , Addgene; plasmid #92280.

Techniques: Imaging

Journal: Current protocols in neuroscience

Article Title: Protocol for assessing neuron-astrocyte spatial interactions using the neuron-astrocyte proximity assay

doi: 10.1002/cpns.91

Figure Lengend Snippet: Donor bleed through and acceptor cross-talk calculations in HEK293T cells. A. The GFP channel images (ex. 488 nm / em. 505 nm) should be stacked from separate FOVs, before stacking the FRET channel images (ex. 488 nm/ em. 615 nm). B. These stacks can then be used to create FOV donor montages of the GFP and FRET channels, these montages can then be stacked in a single file, the donor montage stack. C. From the montage stack we can graph the raw donor bleed through model and by thresholding the PixFRET viewer, we can determine the final donor model. D. For the acceptor controls, the mCherry channel images (ex. 543 nm/ em. 615 nm) should be stacked from separate FOVs, before stacking the FRET channel images (ex. 488 nm/ em. 615 nm). These stacks can then be used to create multiple FOV acceptor montages of the mCherry and FRET channels. E. The mCherry and FRET channel montages can then be stacked in a single file, the acceptor montage stack. F. From the montage stack we can graph the raw acceptor cross-talk model and by thresholding the PixFRET viewing window, we can determine the final acceptor model.

Article Snippet: CMV mCherry GFP BGH , Octeau et al., 2018 , Addgene; plasmid #92280.

Techniques:

Journal: Current protocols in neuroscience

Article Title: Protocol for assessing neuron-astrocyte spatial interactions using the neuron-astrocyte proximity assay

doi: 10.1002/cpns.91

Figure Lengend Snippet: Calculation of donor bleed through and acceptor cross talk in brain slices. A. The GFP channel images (ex. 488 nm/ em. 505 nm) should be stacked from separate FOVs, before stacking the FRET channel images (ex. 488 nm / em. 615 nm). B. These stacks can then be used to create FOV donor montages of the GFP and FRET channels. The channels can then be stacked in a single file, the donor montage stack. C. From the montage stack we can graph the raw donor bleed through model and by thresholding the PixFRET viewer, we can determine the final donor model. D. For the acceptor controls, the mCherry channel images (ex. 543 nm/ em. 615 nm) should be stacked from separate FOVs, before stacking the FRET channel images (ex. 488 nm/ em. 615 nm). These stacks can then be used to create multiple FOV acceptor montages of the mCherry and FRET channels. E. The mCherry and FRET channel montages can then be stacked in a single file, the acceptor montage stack. F. From the montage stack we can graph the raw acceptor cross-talk model and by thresholding the PixFRET viewing window, we can determine the final acceptor model.

Article Snippet: CMV mCherry GFP BGH , Octeau et al., 2018 , Addgene; plasmid #92280.

Techniques:

Journal: Current protocols in neuroscience

Article Title: Protocol for assessing neuron-astrocyte spatial interactions using the neuron-astrocyte proximity assay

doi: 10.1002/cpns.91

Figure Lengend Snippet: Calculation of sensitized emission FRET efficiency in HEK293T cells. A. Example of the image order that should be used in creating the three channel FRET stacks before quantification in PixFRET. B. Representative image of FRET efficiency from the GFP-mCherry fusion experiment. C. Masked image of FRET from panel B, showing the calculated FRET efficiency from individual cells.

Article Snippet: CMV mCherry GFP BGH , Octeau et al., 2018 , Addgene; plasmid #92280.

Techniques:

Journal: Current protocols in neuroscience

Article Title: Protocol for assessing neuron-astrocyte spatial interactions using the neuron-astrocyte proximity assay

doi: 10.1002/cpns.91

Figure Lengend Snippet: Acquisition settings tested to detect FRET in brain slices

Article Snippet: CMV mCherry GFP BGH , Octeau et al., 2018 , Addgene; plasmid #92280.

Techniques:

Journal: Current protocols in neuroscience

Article Title: Protocol for assessing neuron-astrocyte spatial interactions using the neuron-astrocyte proximity assay

doi: 10.1002/cpns.91

Figure Lengend Snippet: FRET efficiency based on tandem GFP-mCherry in HEK293T cells. A. Representative images of the HEK293T cells expressing, GFP alone (top panels), mCherry alone (middle panels) and tandem GFP-mCherry (bottom panels). B. Quantification of FRET efficiency from 20 cells in these three conditions (Mean ± SEM).

Article Snippet: CMV mCherry GFP BGH , Octeau et al., 2018 , Addgene; plasmid #92280.

Techniques: Expressing

Journal: Current protocols in neuroscience

Article Title: Protocol for assessing neuron-astrocyte spatial interactions using the neuron-astrocyte proximity assay

doi: 10.1002/cpns.91

Figure Lengend Snippet: Materials and reagents used for measuring FRET in HEK293T cells

Article Snippet: CMV mCherry GFP BGH , Octeau et al., 2018 , Addgene; plasmid #92280.

Techniques: Transfection, Plasmid Preparation