Abstract
To assess the cytotoxic activity of immune cells, we have developed a51Cr-retention assay in which the radioactivity retained by51Cr-labeled target cells, following coincubation with cytotoxic cells, is monitored using the automated Matrix 96 beta counter. The Matrix 96 is designed for simultaneously counting 96 samples isolated from a 96-well microplate. It uses 96 uniform and independent detectors operating on the principle of avalanche gas ionization in the Geiger-Muller mode. Samples must be dry because the detectors are of the open-window type. Therefore, samples from the 96 wells of the microplate are simultaneously harvested onto a filter using the MicroMate 196, a 96-well cell harvester, dried and quantified in the Matrix 96. Usually the51Cr isotope is measured by the detection of gamma radiation in gamma counters. The Matrix 96, however, monitors Auger electrons, which are also emitted by51Cr. We have shown that the retention assay can be used to monitor the cytotoxic activity of activated lymphocytes including lymphokine-activated killer cells and tumor-infiltrating lymphocytes against various tumor cell lines. This assay is most suitable for experiments in which low E/T ratios are sufficient to detect highly cytotoxic cells, such as clone screening in cloning assays or in limiting-dilution analysis assays. These assays involve processing and reading large numbers of microplates. In this case, the retention assay monitored in the Matrix 96 will improve the work flow and decrease the amount of radioactive waste.
Similar content being viewed by others
References
Albertini MR, Oettel KR, Weil-Hillman G, Lindstrom MJ, Schell K, Hank JA, Sondel PM (1990) Limiting dilution analysis of lymphokine-activated killer cell precursor frequencies in peripheral blood lymphocytes of cancer patients receiving interleukin-2 therapy. J Biol Response Mod 9: 456–462
Brunner KT, Mauel J, Cerottini JC, Chapuis B (1968) Quantitative assay of the lytic action of immune lymphoid cells on51Cr-labeled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14: 181–196
Fisch P, Malkovsky M, Braackman E, Bolhuis RLH, Prieve A, Sosman JA, Lam VA, Sondel PM (1990) Gamma/delta T-cell clones and natural killer-cell clones mediate distinct patterns of non-major histocompatibility complex-restricted cytolysis. J Exp Med 171: 1567–1579
Grimm EA, Mazumder A, Zhang HZ, Rosenberg SA (1982) The lymphokine activated killer cell phenomenon: lysis of NK resistant fresh solid tumor cells by IL-2 activated autologous human peripheral blood lymphocytes. J Exp Med 155: 1823–1941
Hillman GG, Sud S, Dybal EJ, Pontes JE and Haas GP (1992) Combinations of lymphocyte activating agents for expansion of tumor infiltrating lymphocytes from renal cell carcinoma. In: Goldstein AL, Garaci E (eds) Combination therapies: biological response modifiers in the treatment of cancer and infectious diseases. Plenum Press, New York, pp 39–48
Kolber MA, Quinones RR, Gress RE, Henkart PA (1988) Measurement of cytotoxicity by target cell release and retention of the fluorescent dye bis-carboxyethyl-carboxy-fluorescein (BCECF). J Immunol Methods 108: 255–264
Lanier LL, My Le A, Civin CI, Loken MR, Phillips JH (1986) The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J Immunol 136: 4480–4485
Rosenberg SA (1991) Adoptive cellular therapy in patients with advanced cancer: an update. Biol Ther Cancer Updates 1: 1–15
Sharrock CEM, Kaminski E, Man S (1990) Limiting dilution analysis of human T cells: a useful clinical tool. Immunol Today 11: 281–285
Sognier MA, Neft RE, Roe AL, Eberle RL, Belli JA (1991) Dot-blot hybridization: quantitative analysis with direct beta counting. Biotechniques 11: 520–525
Topalian SL, Muul LM, Rosenberg SA (1987) Growth and immunologic characteristics of lymphocytes infiltrating human tumor. Surg Forum 37: 390–391
Weil-Hillman G, Hank JA, Rosenthal NS, Sondel PM (1988) Transient decrease in IL-2 responsive lymphocytes 24 hours after initiation of continuous IL-2 infusion in cancer patients. J Biol Response Mod 7: 424–437
Weil-Hillman G, Fisch P, Prieve AF, Sosman JA, Hank JA, Sondel PM (1989) Lymphokine-activated killer activity induced by in vivo interleukin-2 therapy: predominant role for lymphocytes with increased expression of CD2 and Leu 19 antigens but negative expression of CD16 antigens. Cancer Res 49: 3680–3688
Weil-Hillman G, Schell K, Segal DM, Hank JA, Sosman JA, Sondel PM (1991) Activation of human T cells obtained pre and post IL-2 therapy by anti-CD3 monoclonal antibody plus IL-2: implications for combined in vivo treatment. J Immunother 10: 267–277
Young JDE, Cohn ZA (1988) How killer cells kill. Sci Am 258: 38–44
Author information
Authors and Affiliations
Additional information
This work was supported by the American Cancer Society grant IN-162-C
Rights and permissions
About this article
Cite this article
Hillman, G.G., Roessler, N., Fulbright, R.S. et al. Application of the direct beta counter Matrix 96 for cytotoxic assays: Simultaneous processing and reading of 96 wells using a51Cr-retention assay. Cancer Immunol Immunother 36, 351–356 (1993). https://doi.org/10.1007/BF01742250
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01742250