Team:Goettingen/protocol inVivo

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<div class="proRP">
<div class="proRP">
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<h3 id="general">General information:</h3>
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<h3 id="general">General information:</h3><br />
<table class="table2">
<table class="table2">
<tr><th>Strains for experiments: </th><th> Used peptide:</th></tr>
<tr><th>Strains for experiments: </th><th> Used peptide:</th></tr>
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<tr><td> <i> Candida glabrata</i>:  ATCC2001 (wild type) </td><td> 4, 5</td></tr>
<tr><td> <i> Candida glabrata</i>:  ATCC2001 (wild type) </td><td> 4, 5</td></tr>
<tr><td> <i> Candida glabrata Δssr1</i>:  ATCC2001 (CAGL0H06413g) </td><td> 4</td></tr>
<tr><td> <i> Candida glabrata Δssr1</i>:  ATCC2001 (CAGL0H06413g) </td><td> 4</td></tr>
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</table>
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</table><br />
<p>
<p>
<i>Aspergillus nidulans</i> and<i> Aspergillus fumigatus</i> were washed with 0.96% NaCl and grown in minimal media at 37°C.<br />
<i>Aspergillus nidulans</i> and<i> Aspergillus fumigatus</i> were washed with 0.96% NaCl and grown in minimal media at 37°C.<br />
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<i>Candida glabrata</i> was washed with PBS and grown in YPAD at 30°C.<br />
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<i>Candida glabrata</i> was washed with PBS and grown in YPAD at 30°C.<br /><br />
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</p>
<h3 id="Prod">Production of an <i>A. fumigatus</i> and<i> A. nidulans</i> spore suspension:</h3>
<h3 id="Prod">Production of an <i>A. fumigatus</i> and<i> A. nidulans</i> spore suspension:</h3>
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2. Incubate the plates for 3 days at 37 °C.<br />
2. Incubate the plates for 3 days at 37 °C.<br />
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3.  Afterwards, wash off the spores with 5 ml NaCl-Tween (0.96% NaCl, 0.02% Tween80).<br />
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3.  Afterwards, wash off the spores with 5 ml NaCl-Tween (0.96% NaCl, 0.02% Tween80).<br /><br />
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</p>
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<h3 id="Culti_A">Cultivation of <i>Aspergillus</i> for in vivo experiments:</h3>
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<h3 id="Culti_A">Cultivation of <i>Aspergillus</i> for in vivo experiments:</h3><br />
<p>
<p>
1. Inoculate 50 ml minimal medium in baffeld flasks with 20 µl spores of<i> Aspergillus fumigatus</i> or <i>Aspergillus nidulans.</i>.<br />
1. Inoculate 50 ml minimal medium in baffeld flasks with 20 µl spores of<i> Aspergillus fumigatus</i> or <i>Aspergillus nidulans.</i>.<br />
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2. Incubate the cell suspensions at 37 °C over night on a shaker at least 160 rpm.<br />
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2. Incubate the cell suspensions at 37 °C over night on a shaker at least 160 rpm.<br /><br />
</p>
</p>
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<h3 id="Culti_C">Cultivation of <i>Candida glabrata</i> for<i> in vivo</i> experiments:</h3>
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<h3 id="Culti_C">Cultivation of <i>Candida glabrata</i> for<i> in vivo</i> experiments:</h3><br />
<p>
<p>
1. Inoculate 50 ml YPAD medium with desired strains.<br />
1. Inoculate 50 ml YPAD medium with desired strains.<br />
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2. Incubate the cells at 30 °C over night on a shaker.<br />
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2. Incubate the cells at 30 °C over night on a shaker.<br /><br />
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</p>
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<h3 id="immu">Immunofluorescence Staining </h3>
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<h3 id="immu">Immunofluorescence Staining </h3><br />
<p>
<p>
1. Use a stain expressing the protein of interest and a mutant strain without the protein as negative control (if available) and cultivate them over night (see cultivation of <i>Aspergillus</i>).<br />
1. Use a stain expressing the protein of interest and a mutant strain without the protein as negative control (if available) and cultivate them over night (see cultivation of <i>Aspergillus</i>).<br />
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3. Wash the cells again 3 times with 500 µl NaCl or PBS, centrifuge them at 8000 rpm for 2 minutes and resuspend in 500 µl minimal media or YPAD.<br />
3. Wash the cells again 3 times with 500 µl NaCl or PBS, centrifuge them at 8000 rpm for 2 minutes and resuspend in 500 µl minimal media or YPAD.<br />
3. Add 1 µl StrepMAB Classic Chromeo 488 antibody to the samples and incubated for 1 hour at room temperature in the dark.<br />
3. Add 1 µl StrepMAB Classic Chromeo 488 antibody to the samples and incubated for 1 hour at room temperature in the dark.<br />
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5. Wash 3 times with 500 µl PBS and resuspend in minimal medium or PBS and take pictures with the fluorescence microscope.<br />
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5. Wash 3 times with 500 µl PBS and resuspend in minimal medium or PBS and take pictures with the fluorescence microscope.<br /><br />
</p>
</p>
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<h3 id="Calc">Calcofluor staining</h3>
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<h3 id="Calc">Calcofluor staining</h3><br />
<p>
<p>
1. Add 1 µl Calcofluor stock solution (10mg/ml) to 1 ml cell suspension (after immunofluorescence staining) and incubate for 2 minutes at room temperature.<br />
1. Add 1 µl Calcofluor stock solution (10mg/ml) to 1 ml cell suspension (after immunofluorescence staining) and incubate for 2 minutes at room temperature.<br />
2. Aspirate the staining solution and wash the cells three times in 1 ml PBS. Then resuspend the cells in 500 µl minimal media or YPAD.<br />
2. Aspirate the staining solution and wash the cells three times in 1 ml PBS. Then resuspend the cells in 500 µl minimal media or YPAD.<br />
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3. Analyze the cells with a DAPI filter (365-395 nm) under the fluorescence microscope.<br />
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3. Analyze the cells with a DAPI filter (365-395 nm) under the fluorescence microscope.<br /> <br />
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Latest revision as of 12:56, 17 October 2014

General information:


Strains for experiments: Used peptide:
Aspergillus fumigatus: AfS35 (wild type) 13
Aspergillus nidulans: A4 (wild type) 13
Candida glabrata: ATCC2001 (wild type) 4, 5
Candida glabrata Δssr1: ATCC2001 (CAGL0H06413g) 4

Aspergillus nidulans and Aspergillus fumigatus were washed with 0.96% NaCl and grown in minimal media at 37°C.
Candida glabrata was washed with PBS and grown in YPAD at 30°C.

Production of an A. fumigatus and A. nidulans spore suspension:

1. Plate 10 µl of an A. fumigatus respectively A. nidulans spore suspension were on minimal media plates.
2. Incubate the plates for 3 days at 37 °C.
3. Afterwards, wash off the spores with 5 ml NaCl-Tween (0.96% NaCl, 0.02% Tween80).

Cultivation of Aspergillus for in vivo experiments:


1. Inoculate 50 ml minimal medium in baffeld flasks with 20 µl spores of Aspergillus fumigatus or Aspergillus nidulans..
2. Incubate the cell suspensions at 37 °C over night on a shaker at least 160 rpm.

Cultivation of Candida glabrata for in vivo experiments:


1. Inoculate 50 ml YPAD medium with desired strains.
2. Incubate the cells at 30 °C over night on a shaker.

Immunofluorescence Staining


1. Use a stain expressing the protein of interest and a mutant strain without the protein as negative control (if available) and cultivate them over night (see cultivation of Aspergillus).
2. Centrifuge of 1 ml of each over night culture.
3. Wash the cells 3 times with NaCl or PBS and resuspend them in 500 µl minimal medium or YPAD.
2. Add 20 µl peptide solution (2-3 mg/ml) to the samples and incubate for at least 1 hour 37 °C or at 30°C on a shaker.
3. Wash the cells again 3 times with 500 µl NaCl or PBS, centrifuge them at 8000 rpm for 2 minutes and resuspend in 500 µl minimal media or YPAD.
3. Add 1 µl StrepMAB Classic Chromeo 488 antibody to the samples and incubated for 1 hour at room temperature in the dark.
5. Wash 3 times with 500 µl PBS and resuspend in minimal medium or PBS and take pictures with the fluorescence microscope.

Calcofluor staining


1. Add 1 µl Calcofluor stock solution (10mg/ml) to 1 ml cell suspension (after immunofluorescence staining) and incubate for 2 minutes at room temperature.
2. Aspirate the staining solution and wash the cells three times in 1 ml PBS. Then resuspend the cells in 500 µl minimal media or YPAD.
3. Analyze the cells with a DAPI filter (365-395 nm) under the fluorescence microscope.

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