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In vitro induction and measurement of hemolytic plaque forming cells in man
Abstract:
Following co-cultivation with sheep red cells or ovalbumin, Hypaque-Ficoll-separated human tonsillar lymphocytes were demonstrated to generate specific hemolytic PFC with maximum numbers at day 5-7. PFC were enumerated on poly-L-lysine coupled red cell monolayers in Microtest-II-plates. Plaque formation appeared to be puromycin-sensitive, complement-dependent and showed clear specificity for the antigen present during the inductive culture. Treatment of PFC with mu-chain specific antisera and complement resulted in complete inactivation of PFC; gamma-chain antisera had no effect. The development of such a simple and sensitive assay system permits the analysis of cellular interactions required for the induction of PFC responses in man.
Insights
Human lymphocytes generate specific hemolytic antibody-producing cells (PFC) in response to antigens. This study introduces a sensitive assay to analyze the cellular interactions crucial for inducing these antibody responses in humans.
Area of Science:
- Immunology
- Cell Biology
- Human Physiology
Background:
- Human tonsillar lymphocytes are key players in the adaptive immune response.
- Understanding the induction of antibody-producing cells (PFC) is vital for immune system analysis.
Purpose of the Study:
- To develop and validate a sensitive assay for enumerating human antibody-producing cells.
- To analyze the cellular interactions involved in the induction of PFC responses in humans.
Main Methods:
- Co-cultivation of human tonsillar lymphocytes with sheep red cells or ovalbumin.
- Enumeration of specific hemolytic plaque-forming cells (PFC) using Microtest-II-plates and poly-L-lysine coupled red cell monolayers.
- Assessment of plaque formation sensitivity to puromycin and complement.
- Characterization of PFC using mu-chain and gamma-chain specific antisera.
Main Results:
- Human lymphocytes generated specific hemolytic PFC peaking at 5-7 days post-induction.
- Plaque formation was confirmed to be puromycin-sensitive and complement-dependent.
- PFC exhibited antigen specificity, responding only to the antigen present during culture.
- Treatment with mu-chain specific antisera inactivated PFC, while gamma-chain antisera had no effect, indicating B-cell origin.
Conclusions:
- A simple and sensitive assay for human PFC enumeration was successfully developed.
- The assay allows for the detailed analysis of cellular interactions required for PFC induction.
- The findings highlight the specificity and regulatory mechanisms of human antibody production.