Waiting
Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove

Biology

Preparing T Cell Growth Factor from Rat Splenocytes

doi: 10.3791/402 Published: October 31, 2007

Summary

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

Abstract

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.

Protocol

or Start trial to access full content. Learn more about your institution’s access to JoVE content here
  1. 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.
  2. Spin for 10 min at 4°C to pellet the cells.
  3. Wash the cells twice.
  4. Resuspend the pellet in NH4Cl 0.15 M (5 ml per spleen). Mix gently and continuously with a pipet for 3 min on ice to lyse the erythrocytes. Fill the tube with medium.
  5. Spin for 10 min at 4°C to pellet the cells.
  6. Wash the cells twice.
  7. Count the cells. A rat spleen gives 200-250 million cells.
  8. Seed the cells at 2 million per ml in complete medium: 50 ml per 75 cm2 flask.
  9. Let grow for 48 hours in the incubator.
  10. Spin 15 min at 4°C to pellet the cells.
  11. Collect the supernatant; discard the cells.
  12. Add 15 mg/ml a methyl mannoside to the supernatant. Mix thoroughly.
  13. Filter (0.2 mm)
  14. Aliquot and store at -20°C. Can be kept at 4°C for 10 days if necessary.

Subscription Required. Please recommend JoVE to your librarian.

Discussion

or Start trial to access full content. Learn more about your institution’s access to JoVE content here

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.

Subscription Required. Please recommend JoVE to your librarian.

Disclosures

The authors have nothing to disclose.

Materials

Name Type Company Catalog Number Comments
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
Beta-mercapt–thanol Reagent Sigma-Aldrich M7522
alpha methyl mannoside Reagent Sigma-Aldrich M6882
Concanavalin A 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-mercapt–thanol; 2 ug/ml Concanavalin A.

DOWNLOAD MATERIALS LIST

References

  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. 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).
Preparing T Cell Growth Factor from Rat Splenocytes
Play Video
PDF DOI DOWNLOAD MATERIALS LIST

Cite this Article

Beeton, C., Chandy, K. G. Preparing T Cell Growth Factor from Rat Splenocytes. J. Vis. Exp. (10), e402, doi:10.3791/402 (2007).More

Beeton, C., Chandy, K. G. Preparing T Cell Growth Factor from Rat Splenocytes. J. Vis. Exp. (10), e402, doi:10.3791/402 (2007).

Less
Copy Citation Download Citation Reprints and Permissions
View Video

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
Simple Hit Counter