Effect of antiaggregants on the in vitro viability, cell count and stability of abalone (Haliotis iris) haemocytes

The ability to successfully prepare and preserve haemocyte cells for microscopy and flow cytometry is critical for the investigation of animal immune systems. In this study, we observed the total cell count, in vitro viability and stability of New Zealand black-footed abalone (Haliotis iris) haemocytes with different antiaggregants and handling protocols. Haemocyte stability was evaluated by direct observation of haemocytes under the microscope and calculating the aggregation index. Haemocyte counts and viability were measured via flow cytometry and tested for the effect of different antiaggregants (Alsever's solution at three concentrations, and specialised blood collection tubes containing lithium heparin and K₂EDTA) at different temperatures and storage times. Results showed that Alsever's solution is an effective antiaggregant at haemolymph:antiaggregant dilution ratios of 1:1, 1:2 and 1:3. Lithium heparin was ineffective as an antiaggregant, whereas K₂EDTA was similarly as effective as Alsever's solution. The influence of different mixing techniques (vortex, pipetting and flipping) were subsequently tested using the K₂EDTA Microtainer^® tubes, revealing that proper mixing should be performed immediately. High cell viability can be achieved by mixing samples by either 10 s of vortexing (1000 rpm), 10 times pipetting or 20 times flipping. The in vitro storage of abalone haemocytes in AS and K₂EDTA as antiaggregants at ambient room temperature was highly effective for up to 24 h (75-85% viability; 0.05-0.15 aggregation index) and is recommended for haemocyte studies in H. iris. Utilization of K₂EDTA Microtainer^® tubes were advantageous since they are more cost effective compared to Alsever's solution, and samples can be prepared more efficiently.