Department of Physiology and Biophysics, University of California, Irvine (UCI)
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Beeton, C., Chandy, K. G. Preparing T Cell Growth Factor from Rat Splenocytes. J. Vis. Exp. (10), e402, doi:10.3791/402 (2007).
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. 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 3. 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 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2.
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°C.
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.
The authors have nothing to disclose.
|PBS, 1x, sterile||Reagent||GIBCO, by Life Technologies||14040-182|
|Penicillin / Streptomycin 100x||Reagent||Sigma-Aldrich||P0781||Add 5 ml of 100x solution to 500 ml PBS to prepare PBS-PS|
|Cell strainer, 70 um diameter||Tool||Fisher Scientific||08-771-2|
|Ammonium Chloride (NH4Cl)||Reagent||Sigma-Aldrich||A0171||Prepare a 0.15 M solution in sterile distilled water, keep cold|
|RPMI 1640||Reagent||GIBCO, by Life Technologies||21870-092|
|Fetal Bovine Serum (FCS, FBS)||Reagent||GIBCO, by Life Technologies||16140-071||Heat-inactivated|
|Penicillin / Streptomycin / L-Glutamine||Reagent||Cambrex/BioWhittaker||17-718R||PSG|
|RPMI vitamins, 100x||Reagent||Sigma-Aldrich||R7256|
|Sodium pyruvate, 100x||Reagent||Sigma-Aldrich||S8636|
|Non-essential amino acids, 100x||Reagent||Sigma-Aldrich|
|alpha methyl mannoside||Reagent||Sigma-Aldrich||M6882|
|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.|
1. Beeton C., Barbaria J., Devaux J., Benoliel A.-M., Gola M., Sabatier J.-M., Bernard D., Crest M., Beraud E. Selective blocking of voltage-gated K+ channels treats experimental autoimmune encephalomyelitis and inhibits T-cell activation. J. Immunol. 166:936-944 (2001).
2. Beeton C., Wulff H., Barbaria J., Clot-Faybesse O., Pennington M., Bernard D., Cahalan M.D., Chandy K.G., Beraud E. Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Proc. Natl. Acad. Sci. USA. 98:13942-13947 (2001).
3. Mor, F., Cohen, I.R. Propagation of Lewis rat encephalitogenic T cell lines: T-cell-growth-factor is superior to recombinant IL-2. J. Neuroimmunol. 123: 76-82 (2002).