Isolation, Fixation and Characterization of Juvenile Gilthead Seabream Head Kidney Leukocytes by Flow Cytometry

This protocol standardizes the isolation, fixation, and viability assessment of leukocytes from the head kidney of gilthead seabream by flow cytometry, enabling high-throughput sample processing without compromising quality.

Traditional techniques like manual cell counting with Neubauer chambers and stained blood smears are common in fish immunology. Flow cytometry is becoming popular, but its application in fish studies remains limited. Traditional fish immunology methods are liver-intensive and prone to errors. Juvenile animal studies are limited by challenges in obtaining pure leukocytes and the need for immediate viability assessment, restricting sample throughput.

This protocol enables detailed immune cell analysis by identifying key leukocyte populations and distinguishing live from dead cells. Fixation preserves viability for a month, allowing scalable, flexible, and efficient flow cytometry workflows.

Our findings advance fish immunology by revealing immune mechanisms and environmental impacts on cell viability, aiding the development of improved disease management strategies in aquaculture.

[Narrator] To begin, acclimate juvenile gilthead seabream, Sparus aurata, in recirculating aquaculture systems where water is continuously filtered and recirculated to maintain a stable environment. Ensure that the system includes mechanical and biological filtration, aeration, and temperature control to maintain optimal abiotic parameters. Adjust environmental factors such as photo period, temperature, salinity, pH, and dissolved oxygen levels according to the specific needs of the study. Use a net to gently transfer juvenile gilthead seabream fish into a temporary holding container filled with tank water. After euthanizing the fish, place one fish on its side on a sterile dissection tray. With a scalpel, make a careful incision along the ventral midline of the fish from the vent towards the gills. Use dissection scissors to extend the incision and expose the internal organs. Locate the head kidney, which is situated just behind the gills near the anterior dorsal region of the body cavity, and extends along the top side beneath the vertebral column. With a pair of fine-tipped forceps and scissors, carefully clear away surrounding tissues to better visualize the head kidney. Then lift the head kidney and make precise cuts around it to free it from the surrounding tissues. Immediately place the excised head kidney into a cell strainer positioned within a sterile Petri dish. Pipette two milliliters of Hanks' Balanced Salt Solution, or HBSS, in a sterile Petri dish. Macerate the excised head kidney on the cell strainer using the plunger of a syringe to release the cells into the solution. Next, prepare a density gradient medium solution. Pipette 600 microliters of the density gradient medium solution into five-milliliter polystyrene round-bottomed tubes. Slowly transfer two milliliters of cell suspension from the Petri dish into each tube. Centrifuge at 400g for 45 minutes at four degrees Celsius with the brake turned off. After centrifugation, remove the tube and observe the leukocyte ring. Then use a sterile Pasteur pipette to gently transfer approximately 100 microliters of the leukocyte ring into a two-milliliter microcentrifuge tube. Now, make up the volume to two milliliters with HBSS and gently resuspend the cells. Centrifuge the sample at 400g for 10 minutes at four degrees Celsius. After centrifugation, carefully discard the supernatant without disturbing the pellet at the bottom of the tube. Then, resuspend the purified pellet in one milliliter of HBSS until the final cell concentration is between 10,000 to one million cells per milliliter. Add 50 microliters of dimethyl sulfoxide to a vial containing the reactive dye. After adjusting the cell density to one million cells per milliliter, transfer one milliliter of the cell suspension into two-milliliter microcentrifuge tubes. Prepare viability control samples as given. Induce cell death in tubes three and four by placing them in a water bath at 70 degrees Celsius for seven minutes. To stain the cells, pipette one microliter of the reconstituted fluorescent dye to tubes two and four containing one milliliter of cell suspension and mix well. Incubate the stained tubes at room temperature for 30 minutes, protected from light. Next, wash the cells twice with one milliliter of HBSS, and resuspend the final pellet in 900 microliters of the same buffer. Then pipette 100 microliters of 37% formaldehyde to fix the cells, and incubate at room temperature for 15 minutes. After washing the cells twice with one milliliter of HBSS containing 1% BSA, resuspend in one milliliter of the same solution. Store the samples at four degrees Celsius in a refrigerator for up to one month. Place the fixed sample tube in the flow cytometer sample port for analysis. Record a minimum of 10,000 events for each sample within the singlets gate. Save all data and back it up on external drives or cloud storage. Visualize the flow cytometry data by plotting forward scatter area versus side scatter area to assess cell size and granularity. Perform singlet cell gating and exclude multiplets by plotting forward scatter area verus forward scatter height. Identify the three leukocyte populations based on forward versus side scatter profiles. Set the threshold for viability dye staining in the corresponding fluorescence channel to distinguish live and dead cells. Under optimal conditions, lymphocytes, monocytes, and granulocytes constituted 31%, 38%, and 31% of the leukocyte population respectively, while under thermal stress, lymphocytes decreased to 21.3%, monocytes increased to 45.6%, and granulocytes remained stable at 33.1%. The sample exposed to optimal conditions exhibited higher viability, with a predominance of live cells across all leukocyte populations. In contrast, the thermally stressed sample showed a significant increase in cell death.