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SYTOX Green membrane permeability assay.

WK Wooseong Kim
GZ Guijin Zou
WP Wen Pan
NF Nico Fricke
HF Hammad A. Faizi
SK Soo Min Kim
RK Rajamohammed Khader
SL Silei Li
KL Kiho Lee
IE Iliana Escorba
PV Petia M. Vlahovska
HG Huajian Gao
FA Frederick M. Ausubel
EM Eleftherios Mylonakis
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The pH of phosphate-buffered saline (PBS) was adjusted to the desired values with NaOH or HCl, and then the PBS was filter sterilized through 0.22-μm-pore-size membrane filters. Black, clear-bottom, 96-well plates (Corning no. 3904) were filled with 50 μl of PBS/well containing the indicated concentration of antibiotics. Exponential-phase or stationary-phase antibiotic-tolerant S. aureus MW2 cells prepared as described under “Killing kinetics assay” and “Antibiotic-tolerant MRSA-killing assay,” respectively, were then washed 3 times with the same volume of PBS. The washed cells were adjusted to an OD600 of 0.4 (∼2 × 107 CFU/ml) with PBS. SYTOX Green (Molecular Probes) was added to 10 ml of the diluted bacterial suspension to a final concentration of 5 μM and incubated for 30 min at room temperature in the dark. Fifty microliters of the bacterium-SYTOX Green mixture was added to each well of the 96-well plates containing antibiotics. Fluorescence was measured at room temperature using a spectrophotometer (SpectraMax M2; Molecular Devices) at the excitation of 485 nm and the emission of 525 nm. All experiments were conducted in triplicate.

Copyright and License information:  ©2020 Kim et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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