An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either Piscirickettsia salmonis or IPNV

Peña, Andrea A; Bols, Niels C; Marshall, Sergio H

Piscirickettsia salmonis type strain LF89 ATCC VR 1361 was grown in the cell line CHSE-214 as described previously [19] . Bacteria obtained from the culture supernatant of 15 days post-infection CHSE-214 cells were used to inoculate CHSE-214 and RTS11 cultures in 25 cm 2 plastic tissue culture flasks (Orange) at a concentration of 4.0 × 10 5 cells/ml. Prior to inoculation, 1 ml aliquots from infected CHSE-214 culture were centrifuged for 10 min at 900 × g at 4°C to remove debris. The supernatants were transferred to fresh tubes and centrifuged for 30 min at maximal speed at 4°C to concentrate the bacteria. After the supernatants had been discarded, bacteria pellets were resuspended in the medium appropriate for each cell line. The titre of P. salmonis used in inoculums was 1 × 10 6.8 ml -1 . This titre was determined on CHSE-214 cells and calculated by the method of Reed and Muench [20] . For expression studies, cells were harvested at 2, 5 and 9 days postinfection.