Protocols:

 

Induction of Signal Transduction Pathways

 


Signal transduction pathways are generally induced by addition of e.g. growth factors, interleukins, or other biologically active compounds to the tissue culture medium. As the serum batches used in cell culture contain many growth factors in varying concentrations, it is essential to silence the cells by cultivating them in serum-free or low serum media for 24-48 hours before the experiment. Only after silencing of the cells, phosphorylation-dependent activation of signal transducing proteins can be detected.

IMPORTANT!
In some cases it is very difficult to silence the tissue culture cells once they have been adapted to certain serum batches. Carefully select the sera you are using in your lab. Use experimental setups that do not include washing or centrifugation steps before cell lysis, since these manipulations will lead to rapid activation of MAPK and SAPK signal transduction pathways.

Induction of MAP Kinases and Growth Factor Receptors

General remarks:
We recommend to generally use epithelial islands instead of confluent cell monolayers for induction of MAP Kinases and growth factor receptors. This will result in increased signal strength. For EGF induction of MAP Kinases it is essential to only use cultures consisting of epithelial islands of 5 - 15 cells. Once an epithelial cell layer is being formed, EGF will only poorly activate the MAP Kinase pathway. Note that in the human squamous cell carcinoma cell line A431 EGFR is autoinduced due to overexpression of EGFR.

Cell Culture Conditions:
  1. Seed cells at a density of 105 to 2 x 105 per petri dish (100 mm diameter) in DMEM/10% FCS and grow at 37 C / 7% CO2 until epithelial islands of 5-15 cells have formed.
  2. Wash the cells twice with warm DMEM (without serum, 37C)
  3. Incubate them for 24 to 48 h in serum-free DMEM to starve them.
Induction of MAPK & Growth Factor Receptors:
We strongly recommend to evaluate the individual kinetics for each treatment in the cellular system you use. Time points may vary according to your kinetics. However, peak signals may be obtained between 5 min and 15 min of induction. Concentrations stated below may as well be subject to optimisation in individual assay systems.

a) Negative Control:
no induction

b) Pervanadate Induction:
add 1 mM sodium pervanadate (positive control)

Preparation of orthovanadate:
Adjust a 100 mM solution of sodium orthovanadate to pH 10, the solution is yellow.
Boil the solution until it turns colorless, cool to room temperature.
Readjust to pH 10, boil and cool down until the pH stabilizes at 10 and the solution remains colorless.
Aliquots can be stored at -20C.

Preparation of pervanadate (to be prepared freshly immediately before use):
Dilute 30% H2O2 1:88 to obtain a 100 mM H2O2 solution. Mix equal volumes of both solutions immediately before use. Use at a starting dilution of 1:100; we recommend to evaluate the amount required by titration.

c) FCS Induction:
add 10% FCS
Perform kinetics to evaluate optimal conditions.

d) EGF induction:
add about 10 ng/ml EGF
Perform kinetics to evaluate optimal conditions.
EGF concentration may have to be optimized.


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