Development of adherence techniques for collecting kidney phagocytes from marine fish (Limanda limanda L.)

Protocols have been successfully developed for collecting kidney phagocytes from dab (Limanda limanda L.), based on the adherence of cells to plastic microtitre plates. For comparative purposes, cell adherence was quantified using both total protein measurements (the more rapid method) and counts of lysed adherent cell nuclei. There was a positive correlation (r2=0·67, P<0.01) between the quantity of total protein per well as compared with the numbers of adherent cell nuclei per well. Cell adherence was significantly reduced following the addition of Foetal Calf Serum (FCS) (0·1–10% v/v) to the tissue culture medium (HBSS supplemented with heparin and penicillin/ streptomycin). Adherence was optimal following the addition of 2·5 × 106 kidney cells per well, suspended in FCS-free culture medium. For fish maintained in sea water at 15 ± 1° C, cell adherence after 2 h incubation was highest at 20 ± 1° C, as compared with incubation temperatures of 15 or 25( ± 1)° C. Adherence was also enhanced when using tissue culture grade polystyrene microtitre plates which had been pre-treated by the manufacturer with corona-discharge. Finally, intracellular production of superoxide anion (O2−) was measured in adherent cells using the nitroblue tetrazolium (NBT) assay, whereby O2− production increased in proportion to the number of adherent cells per well. The results provide a baseline for future investigations of intracellular O2− production by the phagocytes of dab and other species of marine flatfish.