Optimization and comparison of the MTT assay and the 3H-TdR assay for the detection of IL-2 in helper T cell precursor assays.
The helper T cell precursor (HTLp) assay is of value for predicting graft-versus-host disease after allogeneic bone marrow transplantation. The assay requires reliable detection of the amount of interleukin 2 (IL-2) produced by one cell. To optimize the IL-2 sensitivity of our HTLp assay we tested an IL-2 dependent cell line, CTLL-2, with two different measurement methods: a colorimetric assay with tetrazolium (MTT) and an isotope incorporation assay with 3H-thymidine (3H-TdR). The test conditions examined encompassed: time without IL-2, preincubation time in IL-2, CTLL-2 cell concentration and different human sera. Due to the different measurement procedures, the volumes of the IL-2 dilutions were 75 microl in assays with MTT and 150 microl in assays with 3H-TdR. We found that it was the amount of IL-2, not the concentration, that limited the growth of CTLL-2 cells. In the most optimal setting the MTT assay could detect 0.6 pg IL-2/well, corresponding to 8 pg/ml. For the 3H-TdR assay the sensitivity was 0.6 pg/well, corresponding to 4 pg/ml. Because of the possibility of IL-2 detection in the whole culture volume (150 microl), we found that the 3H-TdR assay was superior to the MTT assay with a 10-fold better sensitivity in different human sera.
Department of Haematology, Copenhagen University Hospital, Denmark.
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Russell
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Vindelov
D002460:Cell Line; D004396:Coloring Agents / Q000032:analysis*; D005069:Evaluation Studies as Topic; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D007376:Interleukin-2 / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity; D016176:T-Lymphocyte Subsets / Q000737:chemistry*; D006377:T-Lymphocytes, Helper-Inducer; D013778:Tetrazolium Salts / Q000032:analysis*; D013844:Thiazoles / Q000032:analysis*; D013936:Thymidine / Q000032:analysis*; D014316:Tritium / Q000032:analysis
D004396:Coloring Agents; D007376:Interleukin-2; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D014316:Tritium; C022616:thiazolyl blue; D013936:Thymidine