Quantifying FlhA Localization in Bacteria

Strains harboring a chromosomal FlhA-mNeonGreen fusion were grown in LB until reaching an OD₆₀₀ of 0.7–0.8. Cells were harvested, washed in PBS, and applied on 1% agarose pads (Sigma). Slides were imaged using a Nikon Eclipse Ti2 inverted microscope equipped with a sCMOS Prime BSI (Photometrics) camera, a Lumencor Spectra III light engine (Lumencor), and a CFI Plan Apochromat DM ×60 Lambda oil Ph3/1.40 objective (Nikon) using a LED-DA/FI/TR/Cy5/Cy7-A Filter Cube (Semrock). Z-stacks were acquired and the FlhA-mNeonGreen foci were counted using MicrobeJ^{31 (link)}.

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Hüsing S., Halte M., van Look U., Guse A., Gálvez E.J., Charpentier E., Blair D.F., Erhardt M, & Renault T.T. (2021). Control of membrane barrier during bacterial type-III protein secretion. Nature Communications, 12, 3999.

Publication 2021

CFI Plan Apochromat DM ×60 Lambda oil Ph3/1.40 objective

Agarose Cells Chromosomal Light Microscope

Corresponding Organization :

Other organizations : Humboldt-Universität zu Berlin, Max Planck Unit for the Science of Pathogens, University of Utah, Centre National de la Recherche Scientifique, Université de Bordeaux

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Variable analysis

independent variables

Strains harboring a chromosomal FlhA-mNeonGreen fusion

dependent variables

Presence and number of FlhA-mNeonGreen foci

control variables

Growth in LB until reaching an OD600 of 0.7–0.8
Washing cells in PBS
Applying cells on 1% agarose pads (Sigma)
Imaging using a Nikon Eclipse Ti2 inverted microscope equipped with a sCMOS Prime BSI (Photometrics) camera, a Lumencor Spectra III light engine (Lumencor), and a CFI Plan Apochromat DM ×60 Lambda oil Ph3/1.40 objective (Nikon) using a LED-DA/FI/TR/Cy5/Cy7-A Filter Cube (Semrock)

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