Modified cytotoxic T lymphocyte precursor frequency assay by measuring released europium in a time resolved fluorometer.
The frequency of cytotoxic T lymphocyte precursors (CTLpf) can be quantified by using the principle of limiting dilution analysis (LDA). Chromium 51 (51Cr) and europium (Eu) release assays are based on the measurement of marker release after lysis of targets by the effector cells. Although, 51Cr release has been widely used to quantify cell lysis since its introduction, it has several disadvantages such as handling and disposal of radioisotopes as well as health risk to personnel involved performing the assay. This situation has led us to adopt a non-radioactive cytotoxicity assay. After 7 days culture the PHA-stimulated targets are labeled with europium DTPA chelate. Lysis of labeled targets by effectors releases the Eu-DTPA complex in culture medium--a highly fluorescent substance. The amount of fluorescence can be measured in a time resolved fluorometer. We describe here some modifications of the original protocol which include optimising IL-2 requirements, reduction of incubation times, addition of an extra spin before 37 degrees C incubation, readjustment of target cells per volume of labeling buffer and other crucial parameters increasing the specificity and sensitivity of CTLpf assay. We are in agreement with others that the Eu-release assay is specific and reproducible. It can be used for the CTLpf estimation as well as other T cell and non-T cell cytotoxicity assays.
Haque
K
K
University of Bristol Department of Transplantation Sciences, UK.
Truman
C
C
Dittmer
I
I
Laundy
G
G
Denning-Kendall
P
P
Hows
J
J
Feest
T
T
Bradley
B
B
D000328:Adult; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002478:Cells, Cultured; D002614:Chelating Agents / Q000378:metabolism; D003080:Cold Temperature; D003470:Culture Media; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D005063:Europium / Q000378:metabolism*; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000276:immunology*; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects; D004369:Pentetic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D013997:Time Factors
D000900:Anti-Bacterial Agents; D002614:Chelating Agents; D003470:Culture Media; D007376:Interleukin-2; D011994:Recombinant Proteins; D005063:Europium; D004369:Pentetic Acid