Name: clonogenic assay: adherent cells
Uploaded: 2011-03-13T11:00:00
Description: Watch this Scientific Journal Video about Clonogenic Assay: Adherent Cells at JoVE.com

<p class="jove_content">The support of the Australian Institute of Nuclear Science and Engineering is acknowledged. TCK was the recipient of AINSE awards. Epigenomic Medicine Lab is supported by the National Health and Medical Research Council of Australia (566559).  HR is supported by an Australian post-graduate and BakerIDI bright spark awards.  This work is funded by the CRC for Biomedical Imaging Development Ltd (CRC-BID), established and supported under the Australian Government's Cooperative Research Centres program.  CO is the recipient an Australian post-graduate award and a CRC-BID supplementary scholarship.</p>

<p class="jove_title">1. Cell Culture and Experimental Set-up</p>
<ol>
<li>Human keratinocytes are maintained as monolayers in 75 cm<sup>2</sup> tissue culture flasks containing 15 mL of keratinocyte-SFM (K-SFM) medium (GIBCO, serum-free medium) supplemented with L-glutamine (2 mM), epidermal growth factor (5 ng/ mL), bovine pituitary extract (40 &mu;g/ mL) and 20 mg/ mL gentamicin.  Cells are grown in a humidified 5% CO<sub>2</sub> environment at 37&deg;C.</li>
<li>Cells are seeded into 12 x 25 cm<sup>2</sup> tissue culture flasks containing 5...

<ol>
	<li>Puck, T. T., Marcus, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+x-rays+on+mammalian+cells.">Action of x-rays on mammalian cells.</a> <em style='font-style: italic'>J Exp Med</em>. <b>103</b>, 653-666 (1956).</li><li>Hurlin, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+human+papillomavirus+type+18-immortalized+human+keratinocytes+to+a+malignant+phenotype.">Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype.</a> <em style='font-style: italic'>Proc Natl Acad Sci U S A</em>. <b>88</b>, 570-574 (1991).</li></ol>

<p class="jove_content">In this example human FEP-1811 keratinocytes were treated with various concentrations up to 100 &mu;g/ mL CPF; data is shown for 20 &mu;g/ mL CPF for 1 hour at 37&deg;C.  Following treatment cells were irradiated with 4 Gy using a <sup>137</sup>Cs source (Gammacell 1000 Elite irradiator; Nordion International, ON, Canada; 1.6 Gy/min).  For the control untreated and drug only treatments 100 cells were plated in each petri dish and 1000 cells per dish were plated for the irradiated samples.  As indicated in the table above...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Keratinocyte-serum free medium</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>17005042</td>
<td>Supplemented with 2mM L-glutamine, 20&#x03BC;g/ml gentamicin, epidermal growth factor, bovine pituitary extract.
</td>
</tr>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15400-054</td>
<td>0.05% trypsin / EDTA to detach adherent cells; 10 x stock diluted in PBS w/o Ca<sup>2+</sup>/Mg<sup>2+</sup>.</td>
</tr>
<tr>
<td>Dulbecco&#x2019;s modified essential medium</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>11885-084</td>
<td>Supplemented with 10% FBS and 20&mu;g/ml gentamicin; used to neutralise trypsin/EDTA.</td>
</tr>
<tr>
<td>0.09% Saline</td>
<td>Solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td> Used to wash colonies.</td>
</tr>
<tr>
<td>10% Neutral buffered formalin solution</td>
<td>Solution</td>
<td>Sigma-Aldrich</td>
<td>HT501128</td>
<td>~4% formaldehyde; used to fix colonies.</td>
</tr>
<tr>
<td>Crystal violet</td>
<td>Powder</td>
<td>SPI-Chem</td>
<td>02577-MB</td>
<td>0.01% crystal violet solution, prepared in dH<sub>2</sub>O, used to stain colonies.</td>
</tr>
<tr>
<td>Gammacell 1000 elite irradiator</td>
<td>&nbsp;</td>
<td>Nordion International Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes</td>
<td>60 x 15 mm</td>
<td>Falcon</td>
<td>353002</td>
<td>&nbsp;</td>
<tr>
<td>Haemocytometer</td>
<td>&nbsp;</td>
<td>Hawksley; Medical and Laboratory Equipment</td>
<td>AC1000</td>
<td>&nbsp;</td>
<tr>
<td>Cloning box</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Plastic box with holes punched at opposite sides of lid; for storing petri dishes during prolonged incubation for colony formation.</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Type 102</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>Used to count colonies consisting of &gt;50 cells.</td>
</tr>		</tbody>
		</table>
		</div>
	

clonogenic assay: adherent cells

<p class="jove_content">The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956<sup>1</sup>. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture<sup>1</sup>.   Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines.  Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines.  A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line.    Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)<sup>2</sup>. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.</p>

Watch this Scientific Journal Video about Clonogenic Assay: Adherent Cells at JoVE.com

Clonogenic Assay: Adherent Cells

<p class="jove_content">The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years.  Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.</p>

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

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