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Detection of early lymphocyte activation by the fluorescent cell membrane probe N-phenyl-1-naphthylamine
Abstract:
N-phenyl-1-naphthylamine (NPN) becomes fluorescent after binding to hydrophobic regions of cell membranes. Rat and mouse lymphoid cell suspensions stained with NPN showed changes in fluorescence emission 30 min after stimulation with mitogen or antigen, detected by microfluorimetry. Incubation of NPN-labelled mouse and rat thymocytes with phytohaemagglutinin or concanavalin A (Con A) caused an increase in mean cell fluorescence intensity. The response to Con A was inhibited by sodium azide and alpha-methyl mannoside. Stimulation of spleen cells from mice by allogeneic cells, or from tumour-bearing rats by tumour antigen consistently resulted in decreased fluorescence. The 'mixed lymphocyte response' detected only certain genetic differences between mouse strains and was proportional to the ratio of stimulator to responder cell number. The NPN staining procedure offers a simple and rapid assay of immunoreactivity and a means of studying early subcellular changes following lymphocyte activation.
Insights
N-phenyl-1-naphthylamine (NPN) fluorescence detects lymphocyte activation. This method offers a rapid assay for immunoreactivity and early subcellular changes in immune responses.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- N-phenyl-1-naphthylamine (NPN) is a fluorescent probe that binds to hydrophobic regions of cell membranes.
- Lymphocyte activation involves complex cellular and subcellular changes.
Purpose of the Study:
- To evaluate N-phenyl-1-naphthylamine (NPN) as a fluorescent probe for detecting lymphocyte activation.
- To investigate early subcellular changes in lymphocytes following stimulation with mitogens or antigens.
Main Methods:
- Lymphoid cell suspensions from rats and mice were stained with NPN.
- Microfluorimetry was used to detect changes in fluorescence emission after stimulation.
- Specific inhibitors were used to confirm the mechanism of Concanavalin A response.
Main Results:
- NPN fluorescence emission changed within 30 minutes of stimulation with mitogen or antigen.
- Phytohaemagglutinin and Concanavalin A (Con A) increased mean cell fluorescence intensity in thymocytes.
- Con A response was inhibited by sodium azide and alpha-methyl mannoside.
- Allogeneic cell or tumor antigen stimulation of spleen cells decreased fluorescence.
- The mixed lymphocyte response detected specific genetic differences and was proportional to cell ratios.
Conclusions:
- NPN staining provides a simple, rapid assay for immunoreactivity.
- This method can study early subcellular changes during lymphocyte activation.
- NPN fluorescence is a sensitive indicator of immune cell status.