Yeast cells (S. cerevisiae, Y. lipolytica, P. kudriavzevii, P. pastoris and C. apicola) were cultivated at +30°C on YPD plates for 24 h before starting the experiment, Z. lentus was cultivated at +24°C on YPD plates for 48 h. A total of 4 ml of SCD medium in 24-well plate was inoculated to initial optical density of 0.2 (OD600). Three parallel replicates were cultivated for each strain. Cells were cultivated for 18 h (+30°C/+24°C in case of Z. lentus, 800 rpm) followed by centrifugation and resuspension in 200 μl of water. A total of 200 μl of the suspension was transferred to Black Cliniplate (Thermo Fisher Scientific) and mCherry fluorescence was measured with Varioskan (Thermo Electron Corporation). The excitation and the emission wavelengths were 587/610 nm, measurement time was 500 ms using a 12 nm excitation bandwidth. Cell density (OD600) measurement was done for normalizing mCherry fluorescence measurement results. For this, cells were diluted 100× in water, and then OD600 was measured with Varioskan (photometric measurement mode, wavelength = 600 nm, bandwidth = 5 nm, measurement time = 100 ms) using a transparent microtiter plate (Nunc 96F, Thermo Fisher Scientific). Normalization was done by dividing the mCherry measurement value with OD600 result.
Nunc 96f
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunc 96F is a 96-well microplate designed for a variety of laboratory applications. It features a flat-bottom configuration and is made of polystyrene, a commonly used material in laboratory equipment.
Automatically generated - may contain errors
4 protocols using nunc 96f
1
Fluorescent Yeast Strains Cultivation and Analysis
2
Fluorescent Protein Quantification
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Cultivation
samples were collected by centrifugation and resuspended with 200
μL of water. A 200 μL portion of the suspension was transferred
to Black Cliniplate (Thermo Fisher Scientific),, and fluorescence
was measured with Varioskan (Thermo Electron Corporation). The excitation/emission
wavelengths for measurement of BFP (blue fluorescent protein) were
399/456 nm, for Venus (yellow fluorescent protein) 510/530 nm, for
mCherry (red fluorescent protein) 587/610 nm, and for KO2 (orange
fluorescent protein) 550/570 nm, respectively. A 5 nm bandwidth and
500 ms measurement time were used in each measurement. Cell density
(OD600) measurement was done for normalizing fluorescence
measurement results. After fluorescence measurement, cells were diluted
100× into water, and then OD600 was measured with
Varioskan (photometric measurement mode, wavelength = 600 nm, bandwidth
= 5 nm, measurement time = 100 ms) using transparent microtiter plate
(Nunc 96F, Thermo Fisher Scientific). The arbitrary units (AU) reported
in figures were obtained by dividing the fluorescence measurement
value by the OD600 value.
samples were collected by centrifugation and resuspended with 200
μL of water. A 200 μL portion of the suspension was transferred
to Black Cliniplate (Thermo Fisher Scientific),, and fluorescence
was measured with Varioskan (Thermo Electron Corporation). The excitation/emission
wavelengths for measurement of BFP (blue fluorescent protein) were
399/456 nm, for Venus (yellow fluorescent protein) 510/530 nm, for
mCherry (red fluorescent protein) 587/610 nm, and for KO2 (orange
fluorescent protein) 550/570 nm, respectively. A 5 nm bandwidth and
500 ms measurement time were used in each measurement. Cell density
(OD600) measurement was done for normalizing fluorescence
measurement results. After fluorescence measurement, cells were diluted
100× into water, and then OD600 was measured with
Varioskan (photometric measurement mode, wavelength = 600 nm, bandwidth
= 5 nm, measurement time = 100 ms) using transparent microtiter plate
(Nunc 96F, Thermo Fisher Scientific). The arbitrary units (AU) reported
in figures were obtained by dividing the fluorescence measurement
value by the OD600 value.
3
Doxycycline-Induced Gene Expression Analysis
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Cells were cultivated
in duplicate on SCD-LEU-URA with 0, 1, 2, or 5 μg/mL doxycycline
for 20 h (30 °C, 800 rpm). Cells were centrifuged, resuspended
in 200 μL of DDIW, and transferred to Black Cliniplate (Thermo
Fisher Scientific). Venus (yellow fluorescent protein) fluorescence
was measured with Varioskan (Thermo Electron Corporation) using excitation
and emission wavelengths of 510/530 nm (measurement time = 100 ms).
A 100x dilution of the cell suspension was made for OD600 measurement with Varioskan (photometric measurement mode, wavelength
= 600 nm, bandwidth = 5 nm, measurement time = 100 ms) using a transparent
microtiter plate (Nunc 96F, Thermo Fisher Scientific), to normalize
fluorescence measurement originating from different cell densities.
The arbitrary units (AUs) reported in figures were obtained by dividing
the fluorescence measurement value by the OD600 value.
To monitor the effect of DOX on growth, the same cells were used
to inoculate 20 mL of SCD-URA-LEU medium, with 0, 1, 2, or 5 μg/mL
DOX. Cells were incubated at 30 °C at 200 rpm for around 17 h
with OD600 measurements taken regularly.
in duplicate on SCD-LEU-URA with 0, 1, 2, or 5 μg/mL doxycycline
for 20 h (30 °C, 800 rpm). Cells were centrifuged, resuspended
in 200 μL of DDIW, and transferred to Black Cliniplate (Thermo
Fisher Scientific). Venus (yellow fluorescent protein) fluorescence
was measured with Varioskan (Thermo Electron Corporation) using excitation
and emission wavelengths of 510/530 nm (measurement time = 100 ms).
A 100x dilution of the cell suspension was made for OD600 measurement with Varioskan (photometric measurement mode, wavelength
= 600 nm, bandwidth = 5 nm, measurement time = 100 ms) using a transparent
microtiter plate (Nunc 96F, Thermo Fisher Scientific), to normalize
fluorescence measurement originating from different cell densities.
The arbitrary units (AUs) reported in figures were obtained by dividing
the fluorescence measurement value by the OD600 value.
To monitor the effect of DOX on growth, the same cells were used
to inoculate 20 mL of SCD-URA-LEU medium, with 0, 1, 2, or 5 μg/mL
DOX. Cells were incubated at 30 °C at 200 rpm for around 17 h
with OD600 measurements taken regularly.
4
Microtiter Plate Growth Assay
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These studies were performed in 96-well microtiterplates (NUNC-96F, ThermoFisher Scientific, Rochester, NY, USA). Each well contained 100 µL aliquots of fresh MRS or APT media with or without 1 mM H 2 O 2 , and were inoculated from logarithmic phase cultures (i.e., WT and MnKat mutant) growing in either MRS or APT media without H 2 O 2 , to a starting OD 600nm = 0.08-0.09.
Cultures were incubated aerobically at 37 • C with continuous shaking, and changes in OD 600nm were monitored as a function of time using the FLUOStar OPTIMA plate reader system (BMG LABTECH Inc., Durham, NC, USA). Data were plotted as a semi-log plot (OD 600nm vs. time), and maximum specific growth rate (µ max •h -1 ) was calculated from the slope of the line during the exponential phase multiplied by 2.303. The specific growth rate replicates were averaged and fitted to a linear regression model, with SE bars based on small sample size standard deviation formula, using GraphPad Prism 4 for Macintosh (GraphPad Software, San Diego, CA, USA). Data represent the mean of at least two biological replicates.
Cultures were incubated aerobically at 37 • C with continuous shaking, and changes in OD 600nm were monitored as a function of time using the FLUOStar OPTIMA plate reader system (BMG LABTECH Inc., Durham, NC, USA). Data were plotted as a semi-log plot (OD 600nm vs. time), and maximum specific growth rate (µ max •h -1 ) was calculated from the slope of the line during the exponential phase multiplied by 2.303. The specific growth rate replicates were averaged and fitted to a linear regression model, with SE bars based on small sample size standard deviation formula, using GraphPad Prism 4 for Macintosh (GraphPad Software, San Diego, CA, USA). Data represent the mean of at least two biological replicates.
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