Relaxation rate dependence on buffer components Samples were in Carmody buffer and temperature was maintained using a VAHEAT control system.įigure: pH-Countdown temperature dependence We imaged Ni-NTA (cOmplete resin, Roche) agarose beads with bound pH-Countdown in the ' non-disruptive' mode on a DMI8 wide-field microscope controlled with Micro-Manager using a 10x 0.23 NA air objective, a 515/30 nm emission filter, and a SpectraX LED illumination source with 395/25 or 470/24 nm excitation filters.
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Right panel: pH-Countdown or pH-Countdown plus pHlorina traces during yeast growth. Left panel: calibration curve for pH-Countdown. pH-Countdown reveals a decrease from pH ~7.4 to ~6.8 as the culture reaches saturation, which matches the trend reported by pHlorina.įigure: pH-Countdown and pHlorina agree quantitatively in live yeast. Calibration was done in permeabilized yeast in Carmody buffer, pH 6 to 8, with 0.1% digitonin.Ĭytosolic pH in yeast is known to be correlated with growth rate. We measured relaxation ratios in the ' disruptive' regime, with a spontaneous decay interval of 15 seconds. To measure pH-Countdown relaxation ratios and pHlorina fluorescence excitation ratios, we used a Leica DMI8 wide-field microscope with a SOLA light engine, a 40X 0.85 NA objective, a 1.6X mag changer, and DAPI, GFP, TXR cubes, and a custom cube (Semrock FF01-440/40, FF470-Di0, FF01-585/40). 6150 µL of fresh media were inoculated with 50 µL of the overnight growth, and sampled every 75 minutes for OD 600 and fluorescence measurements. cerevisiae BY4741) expressing pH-Countdown from a p416 plasmid and pHlorina (a pH-sensitive, ratiometric red fluorescent protein derived from mApple) from a p413 plasmid were grown in synthetic media without URA or HIS for 24 hours.
Sensor photobleaching per photoactivation-relaxation-photodeactivation measurement cycle was ~1%. time colored horizontal stripes correspond to colored boxes in left panel, showing the relationship between relaxation ratio and pH. Right panel: Calculated whole-cell relaxation ratio vs. Pseudocolor indicates cell-averaged pH (calculated via relaxation ratio), which decreases upon addition of CCCP. Left panel: Timelapse images of mitochondria tagged with pH-Countdown. Therefore we used whole-cell relaxation ratios as a simple estimator of cell-averaged mitochondrial pH, which is robust against motion artifacts.įigure: Mitochondrial pH decrease upon addition of the uncoupler CCCP. Unfortunately for relaxation sensors, the mitochondria move quickly, sometimes changing position by several pixels during our 2 s relaxation interval. These cultured cells are transparent, with almost no detectable scattering or autofluoresence, so they're well suited for dual-color intensity sensors like ratiometric pHluorin or SNARF. The relationship between pH values and relaxation rates at this temperature were calibrated using cells permeabilized with 10 μM nigericin in 200 μL Spexyte buffers (AAT Bioquest) + 300 μL Carmody buffers supplemented with 100 mM KCl at pH 6.5, 7, 7.5 and 8. We induced uncoupling (which is expected to lower mitochondrial pH) by adding a final concentration of 3 μM CCCP. Data was acquired in the ' disruptive' regime with a 2 second decay period after activation. Samples were prepared in 8 well #1.5 glass chambers (Cellvis) and the temperature was maintained using an Okolabs enclosure system. We imaged the samples in live cell imaging solution (ThermoFisher, A14291DJ) + 5 mM glucose at 35☌ on a DMI8 widefield microscope controlled with micromanager using a 63x 1.4 NA oil objective, a 515/30 nm emission filter, and a SpectraX LED illumination source with 395/25 or 470/24 nm excitation filters. We prepared COS7 cells transiently expessing pH-Countdown targeted to the mitochondrial matrix using a COX8A presequence.
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Note that this is a limited PDF or print version animated and interactive figures are disabled. This project is maintained by Maria Ingaramo in the York lab, and was funded by Calico Life Sciences LLC Appendix