Journal: ACS Omega

Article Title: Imaging pH Dynamics Simultaneously in Two Cellular Compartments Using a Ratiometric pH-Sensitive Mutant of mCherry

doi: 10.1021/acsomega.8b00655

Figure Lengend Snippet: mCherryEA reports live-cell pH changes in different cell types. (A) Top: differential interference contrast (DIC) and fluorescence images showing the expression of mCherryEA in the cytosol of Neuro2A cells. Bottom: representative pseudocolored image sequence showing changes in the pixel-by-pixel fluorescence ratio over time in response to a transient NH 4 Cl pulse. (B) The pH response upon exposure to 10 mM NH 4 Cl measured as the F 575nm / F 440nm ratio over time ( n = 20). (C) The pH response in primary astrocytes expressing mCherryEA upon exposure to 10 mM NH 4 Cl ( n = 11).

Article Snippet: Neuro2A and HEK-293 were purchased from ATCC, C57BL/6, and FVB mice were purchased from Charles River Laboratories.

Techniques: Fluorescence, Expressing, Sequencing

Journal: ACS Omega

Article Title: Imaging pH Dynamics Simultaneously in Two Cellular Compartments Using a Ratiometric pH-Sensitive Mutant of mCherry

doi: 10.1021/acsomega.8b00655

Figure Lengend Snippet: In situ pH calibration of mCherryEA in the cytosol and mitochondria live cells. (A) pH titration of Neuro2A cells expressing cytosolic mCherryEA (red line, n = 6, 10 cells each) or ratiometric-pHluorin (green dashed line, n = 3, 10 cells each) using nigericin. (B) pH change upon exposure to a transient 10 mM NH 4 Cl pulse in Neuro2A cells expressing cytosolic mCherryEA that was calibrated using nigericin at the end of the experiment ( n = 4). (C) Example DIC and fluorescence images of a Neuro2A cell showing colocalization of mito-mCherryEA and MitoTracker Deep Red. Cell 1 expressed mito-mCherryEA, but cell 2 was not transfected. Cell 1 shows colocalization (yellow) of mito-mCherryEA (green) and MitoTracker (red). (D) pH titration of Neuro2A cells expressing mito-mCherryEA (red line) and mito-ratiometric-pHluorin (green dashed line) using nigericin plus monensin ( n = 3, 4–15 cells each). (E) pH change upon exposure to a transient 10 mM NH 4 Cl pulse in Neuro2A cells expressing mito-mCherryEA that were calibrated using nigericin plus monensin at the end of the experiment ( n = 7). Bars indicate std.

Article Snippet: Neuro2A and HEK-293 were purchased from ATCC, C57BL/6, and FVB mice were purchased from Charles River Laboratories.

Techniques: In Situ, Titration, Expressing, Fluorescence, Transfection

Journal: ACS Omega

Article Title: Imaging pH Dynamics Simultaneously in Two Cellular Compartments Using a Ratiometric pH-Sensitive Mutant of mCherry

doi: 10.1021/acsomega.8b00655

Figure Lengend Snippet: Metabolic inhibition causes differential acidification in the cytosol vs mitochondria. (A) Representative overlay image showing Neuro2A cells expressing cytosolic ratiometric-pHluorin and mitochondria mCherryEA. (B) Average pH change over time in the cytosol (green, dashed) and mitochondria (red). Treatment with the glycolytic inhibitor 1 mM iodoacetic acid (IAA) caused minor acidification in the cytosol with significant difference in the ratio before and after addition of IAA in individual cells. Although, no acidification was detected in mitochondria (asterisks) for the population, two of the three cells showed significant decrease. Treatment with the mitochondrial inhibitors 5 μM oligomycin and 1 μM FCCP (O/F) caused acidification in both compartments, and in particular, the cytosol exhibited large acidification. (D–F) The metabolism-dependent pH dynamics were independent of the sensor. (D) Representative overlay image showing Neuro2A cells expressing cytosolic mCherryEA and mitochondrial ratiometric-pHluorin. (E, F) Similar pH dynamics were observed when the localization of mCherryEA and ratiometric-pHluorin was switched. The sensors were validated at the end of the experiment by adding 30 mM NH 4 Cl and 10 mM acetic acid ( n = 3, 30 cells total). Error bars are std. Scale bar is 10 μm. mCherryEA (red, λ ex = 575 nm, λ em = 632 nm). ratiometric-pHluorin (green, λ ex = 475 nm, λ em = 525 nm).

Article Snippet: Neuro2A and HEK-293 were purchased from ATCC, C57BL/6, and FVB mice were purchased from Charles River Laboratories.

Techniques: Inhibition, Expressing