Journal: The Journal of General Physiology

Article Title: Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

doi: 10.1085/jgp.201511530

Figure Lengend Snippet: Cell unroofing is useful for a variety of adherent cell types. (A) Cartoon of unroofing approach in which cells are stained with R18 (red) after sonication. (B) Unroofed and stained ND7/23, PC-12, F-11, and HEK293T/17 cells imaged with epifluorescence microscopy. Scale bar applies to all images and is 50 µm.

Article Snippet: To label, patches were perfused with 100–250 nM R18 in recording solution (130 mM NaCl, 3 mM HEPES, and 0.2 mM EDTA, pH 7.2) for 1–2 min. To monitor labeling, patches were excited with epifluorescence (Lambda LS with Smart Shutter; Sutter Instrument) and a 560/10-nm excitation filter (Chroma Technology Corp.) and imaged with a 615/60 emission filter (Chroma Technology Corp.) until fluorescence of the patch reached steady state.

Techniques: Staining, Sonication, Epifluorescence Microscopy

Journal: The Journal of General Physiology

Article Title: Transition metal ion FRET to measure short-range distances at the intracellular surface of the plasma membrane

doi: 10.1085/jgp.201511530

Figure Lengend Snippet: tmFRET between R18 and Co 2+ -C18-NTA in inside-out patches from a Xenopus oocyte. (A) Bright-field and epifluorescence images from a representative patch. Fluorescence images from patches with either 0 or 1 µM Co 2+ before (left) or after (middle) application of C18-NTA and after 20 mM EDTA (right). Scale bar in top left image applies to all images and is 20 µm. (B) Patch fluorescence in Co 2+ was normalized to patch fluorescence in EDTA before and after C18-NTA ( n = 4). Mean (lines) and individual (points) patch values are shown. Asterisk indicates significance at P < 0.05.

Article Snippet: To label, patches were perfused with 100–250 nM R18 in recording solution (130 mM NaCl, 3 mM HEPES, and 0.2 mM EDTA, pH 7.2) for 1–2 min. To monitor labeling, patches were excited with epifluorescence (Lambda LS with Smart Shutter; Sutter Instrument) and a 560/10-nm excitation filter (Chroma Technology Corp.) and imaged with a 615/60 emission filter (Chroma Technology Corp.) until fluorescence of the patch reached steady state.

Techniques: Fluorescence