Difference between revisions of "Team:KU Leuven/Protocols"

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                             <p style="text-align:justify">
 
                             <p style="text-align:justify">
 
                             <h2><b>Medium of HEK 293 cells </b></h2>
 
                             <h2><b>Medium of HEK 293 cells </b></h2>
<br>
+
<h4><b>Medium contents</b></h4>
<b>Medium HEK 293 cells</b>
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<ul>
 
<ul>
 
<li>DMEM (Gibco N° 41965-039) 500 ml<li>
 
<li>DMEM (Gibco N° 41965-039) 500 ml<li>
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<li>10 ml Glutamax (Gibco N° 35050-038) = 10 mM</li>
 
<li>10 ml Glutamax (Gibco N° 35050-038) = 10 mM</li>
 
<li>5 ml Non-Essential-Amino-Acids (Gibco N° 1140-035) = 10x</li>
 
<li>5 ml Non-Essential-Amino-Acids (Gibco N° 1140-035) = 10x</li>
<br>
+
</ul>
<br>                        
+
                     
<b>Growing Conditions</b>
+
<h4><b>Growing Conditions</b></h4>
<br>
+
 
<ul>
 
<ul>
 
<li>Incubate at 37°C and 10% CO<sub>2</sub></li>
 
<li>Incubate at 37°C and 10% CO<sub>2</sub></li>
 
<li>Split cells around 70-90% confluency</li>
 
<li>Split cells around 70-90% confluency</li>
 
<li>Change medium twice a week</li>
 
<li>Change medium twice a week</li>
</ul><br>
+
</ul>
<b><h2>Standard procedure for Trypsinization</b><h2>
+
 
 +
<h2><b>Standard procedure for trypsinization</b></h2>
 +
</br>
 
<ul>
 
<ul>
 
<li>Wash the cells with Versene (Gibco N° 15040-033, ± 2 min)</li>
 
<li>Wash the cells with Versene (Gibco N° 15040-033, ± 2 min)</li>
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<li>Aspirate supernatant and resuspend the cells in medium</li>
 
<li>Aspirate supernatant and resuspend the cells in medium</li>
 
<li>Plate cells for experiments or culture</li>
 
<li>Plate cells for experiments or culture</li>
<br></ul>
+
</ul>
  
<b><h2>Coating of Glass coverslips (for patch-clamp or Ca2+ imaging)</h2></b>
+
<h2><b>Coating of glass coverslips (for patch-clamp or Ca2+ imaging)</b></h2>
<br>
+
<h5><b>Poly-l-lysine (PLL, sigma P2636)</b></h5>
<b>Poly-l-lysine (PLL, sigma P2636)</b>
+
<br>
+
 
<ul>
 
<ul>
 
<li>Dissolved at 0.1 mg/ml in Milli-Q H<sub>2</sub>O</li>
 
<li>Dissolved at 0.1 mg/ml in Milli-Q H<sub>2</sub>O</li>
 
<li>Stored at –20°C</li>
 
<li>Stored at –20°C</li>
 
<li>Once opened stored at RT</li>
 
<li>Once opened stored at RT</li>
</ul><br>
+
</ul>
<b>Coverslips</b>
+
<h5><b>Coverslips</b> (18mm)</h5>
(18mm)
+
 
<ul>
 
<ul>
 
<li>Prepare 12-well plates with a coverslip in each well</li>
 
<li>Prepare 12-well plates with a coverslip in each well</li>
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<li>Add 350 ml of PLL to each coverslip</li>
 
<li>Add 350 ml of PLL to each coverslip</li>
 
<li>Incubate for 20 minutes at RT</li>
 
<li>Incubate for 20 minutes at RT</li>
<li>Aspirate and add Milli-Q H2O to each coverslip</li>
+
<li>Aspirate and add Milli-Q H<sub>2</sub>O to each coverslip</li>
 
<li>Incubate for 20 minutes at RT</li>
 
<li>Incubate for 20 minutes at RT</li>
<li>Aspirate H<sub>2</sub>O and store for experiments</li>
+
<li>Aspirate H<sub>2</sub>O and store for experiments. The plates can be kept for several days at 4°C.</li>
You can keep the plates for several days at 4°C
+
 
</ul>
 
</ul>
<br>
+
<h2><b>Seeding Cells</b></h2>
<b><h2>Seeding Cells</b></h2>
+
 
<b><h4>Seeding cells for single-cell experiments</h4></b>
 
<b><h4>Seeding cells for single-cell experiments</h4></b>
<br>
 
 
<ul>
 
<ul>
 
<li>Spray your hands with ethanol, take the 6 well plate with cells out of the incubator and check the density of the cells</li>
 
<li>Spray your hands with ethanol, take the 6 well plate with cells out of the incubator and check the density of the cells</li>

Revision as of 11:43, 31 August 2017

Protocols

In the lab, we used different experimental procedures. There are protocols for the wet and bacterial lab, for the cell culture lab and for the electrophysiology lab.

Cell Culture

Wet Lab