Abstract
After the successful use of 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) in cell proliferation assays, its use has been established by different workers in cytotoxicity assays and research on leukaemia. In the present study, a colorimetric assay using MTT was adopted to evaluate the cytotoxic activity of chicken intestinal intraepithelial lymphocytes (iIELs), which constitute an important cellular component of the gut-associated lymphoid tissue (GALT). These iIELs are found to exhibit natural killer (NK) cell-like cytotoxic activity, which is spontaneous, non-MHC-restricted, and does not need to be primed. Hitherto, conventional chromium-release assays have been used to evaluate the cytotoxic activity of iIELs, but these assays have disadvantages such as radiation hazards and loss of the cells in washing steps. The mean percentage cytotoxic activity of chicken iIELs evaluated by the colorimetric assay was 90.37±2.53 in a group of 5-week-old chickens and 80.2±3.45 in a group of 8-week-old chickens. These findings established the successful use of a colorimetric assay using MTT for evaluating the cytotoxic activity of chickens iIELs.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- DMEM:
-
Dulbecco's modified Eagle's medium
- DMSO:
-
dimethyl sulphoxide
- E:
-
effector cells
- GALT:
-
gut-associated lymphoid tissue
- GM:
-
growth medium
- iIELs:
-
intestinal intraepithelial lymphocytes
- MTT:
-
3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide
- NK cell:
-
natural killer cell
- OD:
-
optical density
- RPMI:
-
Rosewell Park Memorial Institute medium
- T:
-
target cells
References
Anand Kumar, P., 1995. Immunomodulatory approach for gut immunocompetence in relation to intraepithelial lymphocytes in chicken, (MVSc thesis, Deemed University, Indian Veterinary Research Institute)
Bounous, O.I., Campagnoli, R.P. and Brown, J., 1992. Comparison of MTT colorimetric assay and tritiated thymidine uptake for lymphocyte proliferation assays using chicken splenocytes. Avian Diseases, 36, 1022–1027
Bucy, R.P., Chen, C.L.H., Cihak, J., Losch, U. and Cooper, M.D., 1988. Avian T cells expressing gamma delta receptors localise in the splenic sinusoids and the intestinal epithelium. Journal of Immunology, 141, 2200–2205
Chai, J.Y. and Lillehoj, H.S., 1988. Isolation and functional characterization of chicken intestinal intraepithelial lymphocytes showing natural killer activity against tumour target cells. Immunology, 63, 111–117
Espevik, T. and Meyer, J.N., 1986. A highly sensitive cell line WEHI 164 clone 13 for measuring cytotoxic factor/TNF from human monocytes. Journal of Immunological Methods, 95, 99–105
Govindarajan, R., 1994. Studies on natural killer cell activity in chickens, (MVSc thesis, Deemed University, Indian Veterinary Research Institute)
Heo, D.S., Park, J.G., Hata, K., Day, R., Heberman, R.B. and Whiteside, T.L., 1990. Evaluation of tetrazolium based semi-automatic colorimetric assay for measurement of human antitumour cytotoxicity. Cancer Research, 50, 3681–3690
Hoggenmueller, L., Wakenele, P.S. and Schat, K.A., 1993. Preparation and characterization of chicken intraepithelial leukocytes. Avian Pathology, 22, 509–523
Lillehoj, H.S., 1989. Intestinal intraeptithelial and splenic natural killer cell responses to eimerian infections in inbred chickens. Infection and Immunity, 57, 1879–1884
Lillehoj, H.S. and Chung, K.S., 1992. Postnatal development of T-lymphocyte subpopulations in the intestinal intraepithelium and lamina propria in chickens. Veterinary Immunology and Immunopathology, 31, 347–360
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65, 55–63
Myers, T.J. and Schat, K.A., 1990. Natural killer cell activity of chicken intraepithelial leukocytes against rota virus infected target cells. Veterinary Immunology and Immunopathology, 26, 157–170
Pieters, R, Loonen, A.H., Huismans, D.R., Broekma, G.J., Dirven, M.W.J., Heyenbrok, M.W., Hohlen, K. and Veerman, A.J.P., 1990. In vitro drug sensitivity of cells from children with leukemia using the MTT assay with improved culture conditions. Blood, 76, 2372
Plumb, J.A., Milroy, R. and Kaye, S.B., 1989. Effects of the pH dependence of 3-(4,5-dimethyl thiazol)-2,5-diphenyl-tetrazolium based assay. Cancer Research, 49, 4435–4440
Twentyman, P.R. and Luscombe, M., 1987. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. British Journal of Cancer, 56, 279
Van de Loosdrecht, A.A., Nennie, E., Ossenkoppele, G.J., Beelen, R.H.J. and Langenhuijsen, M.M.A.C., 1991. Cell mediated cytotoxicity against U937 cells by human monocytes and macrophages in a modified colorimetric MTT assay. A methodological study. Journal of Immunological Methods, 141, 15–22
Van de Loosdrecht, A.A., Beeleen, R.H.J., Ossankoppele, G.J., Broekhoven, M.G. and Langenheijsen, M.M.A.C., 1994. A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. Journal of Immunological Methods, 174, 311–320
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kumar, P.A., Das, S.K. A colorimetric assay to evaluate the cytotoxic activity of the intestinal intraepithelial lymphocytes of chickens. Veterinary Research Communications 20, 513–518 (1996). https://doi.org/10.1007/BF00396294
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00396294