The cellular death pattern of primary haemocytes isolated from the black tiger shrimp (Penaeus monodon)

• The good quality of primary haemocytes is found in the first 48 h of culture.
• Z-VAD–FMK could not completely stop primary haemocytes dying through apoptosis.
• NaF induces numbers of primary haemocytes to die via the apoptotic mechanism.

A key to successfully generate the penaeid shrimp cell line is to find out how primary cells died. The most suitable period to culture Penaeus monodon haemocytes was in the first 48 h of culture because cells had normal morphology, high percent of viable cells (65.29 ± 5.43%), low percent of early (11.75 ± 1.30%) and late apoptotic cells (15.47 ± 11.71%) determined by Annexin V and TUNEL including constant IAP (0.06 ± 0.01–0.07 ± 0.01) and caspase-3 expression (0.30 ± 0.06–0.39 ± 0.10) by real-time PCR throughout the experiment. Moreover, adding 50 and 250 μM of the cell permeable pan caspase inhibitor Z-VAD–FMK produced some melanised cells since the 48th hour, while percent of viable cells was decreased since the 24th hour with no difference in percent of early and late apoptotic cells compared to control at each time point. No difference of IAP and caspase-3 expression level in both Z-VAD–FMK groups was found compared to control and vehicle groups at each time point, excluding caspase-3 in 250 μM Z-VAD–FMK at the 24th hour was higher than control and vehicle. Supplementing sodium fluoride (NaF) induced cell membrane damage and cellular shrinkage of primary haemocytes within 2 h. Even percent of viable cells was reduced down to zero and percent of late apoptotic cells was increased by 2 h of incubation in 25 and 50 mM NaF, IAP and caspase-3 in all NaF groups was not different from control. These results indicate that a number of primary haemocytes derived in this study die through the apoptotic process.