TY - JOUR
T1 - Factors influencing in vitro respiratory burst assays with head kidney leucocytes from rainbow trout, Oncorhynchus mykiss (Walbaum)
AU - Chettri, Jiwan Kumar
AU - Holten-Andersen, Lars
AU - Buchmann, Kurt
PY - 2010/7
Y1 - 2010/7
N2 - Head kidney leucocytes are central elements in a number of in vivo and in vitro assays elucidating innate and adaptive immune mechanisms in teleosts following stimulation with various antigens. These systems are sensitive to several factors affecting the outcome of the assays. The present work describes the importance of temperature, cell concentration, exposure time and immune-modulatory molecules on the respiratory burst activity (RBA) of rainbow trout head kidney leucocytes in vitro. Some variation in RBA was observed among individual fish. However, use of cells pooled from four individuals produced satisfactory results following exposure to phorbol 12-myristate 13-acetate, zymosan and β-glucan. Temperature was shown to have a significant effect on production of reactive radicals as illustrated by a high activity in cells maintained at 15-20 °C and a reduced activity at temperature extremes (1, 4 and 30 °C). Highest activity was found at a cell concentration of 1 × 107 cells mL-1. Reactivity showed a clear decline when cells were exposed for more than 4 h. Moreover, incubation of cells with inhibitory substances viz., DiMePE2, cortisol and superoxide dismutase decreased the RBA. It is concluded that several biotic and abiotic factors should be taken into account when conducting RBA assays with head kidney leucocytes for elucidation of rainbow trout immune responses.
AB - Head kidney leucocytes are central elements in a number of in vivo and in vitro assays elucidating innate and adaptive immune mechanisms in teleosts following stimulation with various antigens. These systems are sensitive to several factors affecting the outcome of the assays. The present work describes the importance of temperature, cell concentration, exposure time and immune-modulatory molecules on the respiratory burst activity (RBA) of rainbow trout head kidney leucocytes in vitro. Some variation in RBA was observed among individual fish. However, use of cells pooled from four individuals produced satisfactory results following exposure to phorbol 12-myristate 13-acetate, zymosan and β-glucan. Temperature was shown to have a significant effect on production of reactive radicals as illustrated by a high activity in cells maintained at 15-20 °C and a reduced activity at temperature extremes (1, 4 and 30 °C). Highest activity was found at a cell concentration of 1 × 107 cells mL-1. Reactivity showed a clear decline when cells were exposed for more than 4 h. Moreover, incubation of cells with inhibitory substances viz., DiMePE2, cortisol and superoxide dismutase decreased the RBA. It is concluded that several biotic and abiotic factors should be taken into account when conducting RBA assays with head kidney leucocytes for elucidation of rainbow trout immune responses.
KW - Former LIFE faculty
KW - head kidney leucocytes
KW - immunostimulant
KW - rainbow trout
KW - respiratory burst activity
KW - temperature
U2 - 10.1111/j.1365-2761.2010.01160.x
DO - 10.1111/j.1365-2761.2010.01160.x
M3 - Journal article
C2 - 20384908
SN - 0140-7775
VL - 33
SP - 593
EP - 602
JO - Journal of Fish Diseases
JF - Journal of Fish Diseases
IS - 7
ER -