Here, we present the details of the experimental procedure used to analyze cytokine-induced growth arrest of different cancer cells in vitro.
Method Article
Analyzing cytokine-induced growth arrest
https://doi.org/10.1038/protex.2013.020
This work is licensed under a CC BY-NC 3.0 License
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
You are reading this latest protocol version
Here, we present the details of the experimental procedure used to analyze cytokine-induced growth arrest of different cancer cells in vitro.
The most stringent criterion defining cellular senescence is stable and permanent growth arrest in the absence of the inducing agent. Thus, we developed a method to first treat the cells with defined combinations of cytokines, then remove the cytokines and further cultivate the cells in the absence of cytokines.
Human cancer cell lines from the NCI 60 panel.
Human IFN-gamma (R&D Systems, Wiesbaden, Germany).
Human TNF (R&D Systems).
Trypan Blue Stain 0.4% from Gibco (Life Technologies; Darmstadt, Germany).
MultiscreenTM HTS 96 well Filtration Plates (Millipore, Billerica, MA, USA).
BrdU-based Cell Proliferation ELISA (Roche Diagnostics, Mannheim, Germany).
Vector®SG Substrate Kit for Peroxidase from Vector Laboratories (Burlingame, CA, USA).
Zeiss Axiovert 25 microscope (Zeiss, Oberkochen, Germany).
Neubauer counting-chamber (Karl Hecht GmbH, Sondheim, Germany).
ELISPOT reader (Bioreader®-3000; BIO-SYS, Karben, Germany).
The overall procedure takes about 2 - 3 weeks.
Ensure that at the beginning of cytokine treatment, the cells are 5 - 25% confluent.
The duration of the treatment depends on the growth kinetics of the cancer cells (incubate for approx. two duplication times).
The cytokine sensitivity of the different cancer cell lines may differ over a broad concentration range.
Toxic side effects of the cytokines have to be evaluated beforehand.
A cell line that is driven into senescence should show complete growth arrest after removal of the cytokines, i.e. there should not be any increase of the cell number of the cytokine-treated cells.
We declare that there may be a potential financial interest as far as cancer treatment by cytokine-induced senescence is concerned.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
You are reading this latest protocol version