Name: preparing t cell growth factor from rat splenocytes
Uploaded: 2007-10-31T22:24:33
Description: Watch this Scientific Journal Video about Preparing T Cell Growth Factor from Rat Splenocytes at JoVE.com

<ol>
<li>Take Lewis rat spleens (rats between 160-200g are best). Dilacerate on ice in a petri dish containing PBS + antibiotics (PBS-PS) using a cell strainer. Put in a 50 ml tube. Fill with PBS-PS.</li>
<li>Spin for 10 min at 4&deg;C to pellet the cells.</li>
<li>Wash the cells twice.</li>
<li>Resuspend the pellet in NH<sub>4</sub>Cl 0.15 M (5 ml per spleen). Mix gently and continuously with a pipet for <strong>3 min</strong> on ice to lyse the erythrocytes. Fill the tube with medium.</li>
<li>Spin for 10 min at 4&deg;C to pellet the cells.</...

<ol>
	<li>Beeton, C., Barbaria, J., Devaux, J., Benoliel, A. -. M., Gola, M., Sabatier, J. -. M., Bernard, D., Crest, M., Beraud, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+blocking+of+voltage-gated+K++channels+treats+experimental+autoimmune+encephalomyelitis+and+inhibits+T-cell+activation.">Selective blocking of voltage-gated K+ channels treats experimental autoimmune encephalomyelitis and inhibits T-cell activation.</a> <em style='font-style: italic'>J. Immunol</em>. <b>166</b>, 936-944 (2001).</li><li>Beeton, C., Wulff, H., Barbaria, J., Clot-Faybesse, O., Pennington, M., Bernard, D., Cahalan, M. D., Chandy, K. G., Beraud, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+blockade+of+T+lymphocyte+K++channels+ameliorates+experimental+autoimmune+encephalomyelitis,+a+model+for+multiple+sclerosis.">Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis.</a> <em style='font-style: italic'>Proc. Natl. Acad</em>. <b>98</b>, 13942-13947 (2001).</li><li>Mor, F., Cohen, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+Lewis+rat+encephalitogenic+T+cell+lines:+T-cell-growth-factor+is+superior+to+recombinant+IL-2.">Propagation of Lewis rat encephalitogenic T cell lines: T-cell-growth-factor is superior to recombinant IL-2.</a> <em style='font-style: italic'>J. Neuroimmunol</em>. <b>123</b>, 76-82 (2002).</li></ol>

<p class="jove_content">We prepare TCGF from Lewis rat splenocytes since we regularly euthanize naive rats from this strain to harvest serum and thymi to stimulate Lewis rat T cell lines in vitro. This TCGF can be used to promote the growth and survival of T cell lines from other rat strains. TCGF can also be prepared from other strains of rats.</p>

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		<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>PBS, 1x, sterile</td>
<td>Reagent</td>
<td>Gibco / Invitrogen</td>
<td>14040-182</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin / Streptomycin 100x</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P0781</td>
<td>Add 5 ml of 100x solution to 500 ml PBS to prepare PBS-PS</td>
</tr>
<tr>
<td>Cell strainer, 70 um diameter</td>
<td>Tool</td>
<td>Fisher</td>
<td>08-771-2</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Chloride (NH4Cl)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A0171</td>
<td>Prepare a 0.15 M solution in sterile distilled water, keep cold</td>
</tr>
<tr>
<td>RPMI 1640</td>
<td>Reagent</td>
<td>Gibco / Invitrogen</td>
<td>21870-092</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (FCS, FBS)</td>
<td>Reagent</td>
<td>Gibco / Invitrogen</td>
<td>16140-071</td>
<td>Heat-inactivated</td>
</tr>
<tr>
<td>Penicillin / Streptomycin / L-Glutamine</td>
<td>Reagent</td>
<td>Cambrex / Biowhittaker</td>
<td>17-718R</td>
<td>PSG</td>
</tr>
<tr>
<td>RPMI vitamins, 100x</td>
<td>Reagent</td>
<td>Sigma</td>
<td>R7256</td>
<td>&nbsp;</td>
<tr>
<td>Sodium pyruvate, 100x</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S8636</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential amino acids, 100x</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Beta-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M7522</td>
<td>&nbsp;</td>
<tr>
<td>alpha methyl mannoside </td>
<td>Reagent</td>
<td>Sigma</td>
<td>M6882</td>
<td>&nbsp;</td>
<tr>
<td>Concanavalin A</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M6882</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			To prepare complete culture medium add the following to a 500 ml bottle of RPMI 1640 and sterile-filter: 10% FCS; 1 bottle of PSG; 5 ml RPMI vitamins; 5 ml sodium pyruvate; 5 ml non-essential amino acids; 50 uM beta-mercaptoethanol; 2 ug/ml Concanavalin A.		</div>
		

preparing t cell growth factor from rat splenocytes

<p class="jove_content">Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion <sup>1,2</sup>. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization <sup>3</sup>.  Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro <sup>3</sup>. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. </p>
<p class="jove_content">Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium.  The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.</p>

Watch this Scientific Journal Video about Preparing T Cell Growth Factor from Rat Splenocytes at JoVE.com

Preparing T Cell Growth Factor from Rat Splenocytes

<p class="jove_content">We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.</p>

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

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