Project Details
Description
After cultured with gentamicin, vancomycin, linezolid, meropenem and ciprofloxacin for 24 hours, the respiration of the cells was compared with that of the blank control cells, and the cells were stained with mitosox and TMRM by flow cytometry. Mitochondrial oxygen consumption rate will be assessed with Seahorse XFe 96 Extracellular Flux
Analyzer for plated cells, and an Oroboros O2K for cells in suspension. Additionally, glycolytic
activity (media acidification) will be performed with the Seahorse and production of reactive
oxygen species (ROS) will be performed using the LED2-module on the Oroboros. Fluorescent microscopy using a Nikon TIRF/STORM microscope will be used to assess mitochondrial structure
and organization, and measure membrane potential (with the negatively charged probe TMRM).
Additionally, ATP levels in cells will be measured using the ATPlite Assay kit (luciferase -based in a
96 well plate and fitting to a standard curve) and the luminescence reader VICTOR Nivo (both
PerkinElmer, Waltham, USA).
Analyzer for plated cells, and an Oroboros O2K for cells in suspension. Additionally, glycolytic
activity (media acidification) will be performed with the Seahorse and production of reactive
oxygen species (ROS) will be performed using the LED2-module on the Oroboros. Fluorescent microscopy using a Nikon TIRF/STORM microscope will be used to assess mitochondrial structure
and organization, and measure membrane potential (with the negatively charged probe TMRM).
Additionally, ATP levels in cells will be measured using the ATPlite Assay kit (luciferase -based in a
96 well plate and fitting to a standard curve) and the luminescence reader VICTOR Nivo (both
PerkinElmer, Waltham, USA).
| Status | Not started |
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Free keywords
- mitochondria
- Antibiotics