A rapid colorimetric assay for the determination of IL-2-producing helper T cell frequencies.
Interleukin 2 (IL-2) activity is tested in conditioned media by assessing its ability to support proliferation of selected IL-2 dependent T cell lines, conventionally measured by [3H]thymidine incorporation. Here, we compare this [3H]thymidine uptake test for measuring IL-2 activity with a rapid and sensitive colorimetric method which is based on the ability of viable cells to cleave 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The sensitivity of the colorimetric method was dependent on the indicator cell line used, being greatest with the cytotoxic T cell line 16 (CTLL-16). The colorimetric method is at least as sensitive as [3H]thymidine uptake tests, does not rely on radioactivity, and is ideally suited to screen large numbers of individual samples for IL-2 activity. The latter point was demonstrated by calculating IL-2-producing helper T cell frequencies in heterogeneous murine lymphocyte populations: in this assay, splenic T cells were clonally expanded under limiting dilution conditions and supernatants conditioned by these in vitro growing T cell clones were tested for IL-2 activity with the colorimetric method. This allowed us to obtain reliable estimates of the frequency of progenitor cells of IL-2-producing T cell clones in various populations.
Heeg
K
K
Reimann
J
J
Kabelitz
D
D
Hardt
C
C
Wagner
H
H
D000818:Animals; D003124:Colorimetry; D007376:Interleukin-2 / Q000096:biosynthesis*; D007958:Leukocyte Count; D051379:Mice; D008808:Mice, Inbred CBA; D013601:T-Lymphocytes / Q000145:classification; D006377:T-Lymphocytes, Helper-Inducer / Q000378:metabolism*; D013778:Tetrazolium Salts; D013844:Thiazoles
D007376:Interleukin-2; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue