A colorimetric assay for the quantification of brook trout (Salvelinus fontinalis) lymphocyte mitogenesis

Flow cytometry as a tool for assessment of the fish cellular immune response to pathogens

“…More recently, an alternative colorimetric method has been described (Daly et al, 1995). FCM assays of lymphocyte proliferation are routinely used in humans for diagnostic and clinical investigations.…”

Mitogen inducedin vitrostimulation of lymphoid cells from organs of sea bass (Dicentrarchus labraxL.)

“…Notwithstanding this, the results reveal 228 M. GALEOTTI ET AL. that in some cases the in vitro mitogenic responses of leucocytes of di#erent fish species vary with the type of mitogen used. Daly et al (1995), using a similar method of detection, found that brook trout PBL responded more vigorously to LPS than to PHA-P and Con-A. Reitan and Thuvander (1991) observed that in both Atlantic salmon and rainbow trout, PBL and spleen leucocytes tended to respond better to LPS than to PHA-P.…”

“…During the last 6 years, while studying the immunoreactions of fish, various researchers have optimised the in vitro lymphocyte stimulation test (Luft et al, 1991;Faisal & Hargis, 1992;Hardie et al, 1993;Ahne, 1994;Hamers, 1994;Daly et al, 1995). The conditions for the culture and stimulation of lymphoid cells of di#erent fish vary slightly; consequently, the study of each new species requires, in most cases, a modification of reported methods.…”

Cytokine-mediated antimicrobial immune response of catfish,Clarias gariepinus, as a defence againstAeromonas hydrophila

“…The mixture was incubated for 3 h at 25 C before the cultures were supplemented with 10% heat-inactivated FCS and 5% heat-inactivated homologous fish plasma. The colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay described by Daly et al (1995) was used to determine the proliferation of HK leucocytes. After 48 h culture, 20 l of MTT (5 mg ml 1 PBS) was added to each well of the leucocyte cultures and incubated for another 4 h at 25 C. The tissue culture plates were centrifuged at 500 g for 10 min, and the supernatant fluid was then carefully removed without disturbing the cell pellet or precipitate.…”