A flow cytometric (FCM) assay for detection of immunomodulatory effects of environmental factors on the humoral response of @entity11806 ( @entity11806 ) is described and validated. This technique combines exposure of whole animals or leucocyte cultures to immunomodulatory agents/conditions with in vitro mitogenic activation of B-lymphocytes. The proportion of leucocytes undergoing blastogenesis following in vitro stimulation with lipopolysaccharide (LPS) is quantified by FCM analysis of forward and side scatter properties. In addition, binding of a @entity2822 labelled anti-rainbow trout immunoglobulin M monoclonal antibody (anti-RBT SIgM- @entity2823 ), quantified by FCM analysis, is used to determine the ability of the lymphoblasts to express surface immunoglobulin M (SIgM). Through a series of calibration steps, it was confirmed that anti-RBT IgM- @entity2823 was specific for B-lymphocyte SIgM in @entity11806 . Binding of anti-RBT IgM- @entity2823 to @entity11806 SIgM positive leucocytes was effectively blocked with salmon serum and an isotype control was established. B-lymphocytes were partially removed from a population of leucocytes through adherence to a nylon wool column, which then demonstrated a consequent reduction in anti-RBT IgM- @entity2823 binding. Using anti-RBT IgM- @entity2823 as a marker, the distribution of resting lymphocytes expressing SIgM in lymphoid tissues of juvenile @entity11806 was described. The mean percentage of SIgM positive cells in spleen, pronephros and blood were found to be 62.1 (+/-2.82), 34.8 (+/-1.86) and 56.7% (+/-4.7) of all viable leucocytes, respectively. In a time-course experiment for optimal in vitro activation of leucocytes for this assay, blastogenesis and up-regulation of SIgM expression of splenic leucocytes were observed through FCM by 4 days post in vitro stimulation with LPS, continued through 7 days, but was no longer visible by 10 days post stimulation. Using this assay, reduced expression of SIgM in splenic and pronephric B-lymphocytes was detected following in vitro exposure to physiologically relevant stress concentrations of @entity701 in conjunction with mitogenic stimulation. This technique will be a useful addition to the assays already available in the rapidly growing field of fish immunology.

In vitro detection of functional humoral immunocompetence in juvenile XXXX ( @entity11806 ) using flow cytometry.

@entity2823 :: ('MESH:D016650', 'Chemical') :: ['FITC'] @entity2822 :: ('CHEBI:37926', 'Chemical') :: ['fluorescein isothiocyanate'] @entity11806 :: ('74940', 'Species') :: ['Oncorhynchus tshawytscha', 'chinook salmon'] @entity701 :: ('MESH:D006854', 'Chemical') :: ['cortisol']
@entity11806 :: (74940,Species) :: ['chinook salmon']
