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Agar plating technique for enumeration of IL-2 producing cells in human peripheral blood mononuclear leukocytes
C Hirunpetcharat1, W Kasinrerk, S Makonkawkeyoon
1Section of Microbiology and Immunology, Faculty of Associated Medical Sciences, Chiang Mai University, Thailand.
Abstract:
An agar plating technique for detection and enumeration of IL-2 producing cells from human peripheral blood mononuclear leukocytes (PBML) has been developed. This method is based on the principle that PHA-stimulated PBML, as effector cells, secrete interleukin 2 (IL-2) into soft agar containing mouse 3-day Con A blasts as IL-2 dependent responder cells. The IL-2 dependent Con A blasts proliferating around the IL-2 producing cells form colonies or clusters of cells and are easily visualized under a dissecting microscope. The development of IL-2 producing cells was optimum when 1 X 10(6) cells/ml PBML were stimulated with 2 micrograms/ml PHA-P for 4 hours, and when 2.5 X 10(5) cells were co-cultured with 6 X 10(6) Con A blasts in soft agar for 5 days. The average number of IL-2 producing cells in 10 normal healthy controls were 754 +/- 94 (mean +/- S.E.M.) cells/10(6) PBML. The numbers of IL-2 producing cells and the levels of IL-2 produced were highly correlated (r = 0.929). The subpopulation of lymphocytes in the colonies was shown to be mostly murine T-cells, since they were mostly Thy 1.2 positive, CD3 negative and surface immunoglobulin negative. This technique is very simple to perform and provides an accurate and straightforward means to enumerate IL-2 producing cells from human PBML in a variety of human immunologic disorders.
Insights
A new agar plating method accurately counts interleukin-2 (IL-2) producing cells in human blood. This technique aids in studying immune disorders by quantifying IL-2 secreting cells.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Interleukin-2 (IL-2) is crucial for immune responses.
- Quantifying IL-2 producing cells is vital for understanding immune function and disorders.
- Existing methods for enumeration can be complex or lack sensitivity.
Purpose of the Study:
- To develop and validate a simple, accurate agar plating technique for detecting and enumerating IL-2 producing cells from human peripheral blood mononuclear leukocytes (PBML).
- To optimize conditions for IL-2 producing cell development and visualization.
- To establish baseline values in healthy individuals and assess correlation with IL-2 levels.
Main Methods:
- Developed an agar plating assay using PHA-stimulated human PBML as effector cells and mouse Con A blasts as IL-2 dependent responder cells.
- Optimized PBML stimulation (cell concentration, PHA-P concentration, incubation time) and co-culture conditions (PBML:blast ratio, agar incubation time).
- Visualized and enumerated IL-2 producing cells by counting colonies formed by proliferating Con A blasts under a dissecting microscope.
Main Results:
- Optimal conditions involved 1 x 10(6) cells/mL PBML stimulated with 2 µg/mL PHA-P for 4 hours, co-cultured with 6 x 10(6) Con A blasts in soft agar for 5 days.
- The average number of IL-2 producing cells in healthy controls was 754 ± 94 cells/10(6) PBML.
- A high correlation (r = 0.929) was observed between the number of IL-2 producing cells and IL-2 levels. Colonies consisted mainly of murine T-cells (Thy 1.2 positive, CD3 negative).
Conclusions:
- The developed agar plating technique is simple, accurate, and straightforward for enumerating IL-2 producing cells from human PBML.
- This method can be applied to study immune responses in various human immunologic disorders.
- The technique provides a reliable measure of cellular IL-2 production, correlating well with cytokine levels.
