A comparison of assays for the response of primary human T-cells upon stimulation with interleukin-2, interleukin-4 and interleukin-7.
The most commonly used assay to quantitate the response of peripheral T-cells upon stimulation with growth factors is determination of incorporated [3H]TdR. We compared this test to three other methods: 1. direct counting of cells with a Coulter type counter as reference assay, 2. a colorimetric assay using the tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium (MTT), which is a cheap and increasingly popular non-radioactive method and 3. incorporation of the thymidine analog 5-bromo-2'-deoxyuridine detection with a monoclonal antibody on cytospins. Primary human PHA-blasts from greater than 30 healthy individuals were stimulated with IL-2, IL-4 and IL-7 and assayed with up to four different methods. We discuss the advantages and disadvantages of the assays used and the effects of differences between cell preparations. We observed no significant variations between individuals for the dose dependence, but the relative efficiency of IL-4 compared to IL-2 and IL-7 was variable. This was probably due to the slower response observed upon stimulation with this factor.
Duschl
A
A
Theodor-Boveri-Institüt fur Biowissenschaften Universität Würzburg, FRG.
Jahn
U
U
Bertling
C
C
Sebald
W
W
D000911:Antibodies, Monoclonal; D001973:Bromodeoxyuridine; D002455:Cell Division / Q000187:drug effects; D004396:Coloring Agents; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D015847:Interleukin-4 / Q000494:pharmacology*; D015851:Interleukin-7 / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects*; D013778:Tetrazolium Salts; D013844:Thiazoles
D000911:Antibodies, Monoclonal; D004396:Coloring Agents; D007376:Interleukin-2; D015851:Interleukin-7; D013778:Tetrazolium Salts; D013844:Thiazoles; D015847:Interleukin-4; C022616:thiazolyl blue; D001973:Bromodeoxyuridine