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문제 정의
- , 1983) and lymphoid (Tatner and Findly, 1991) organ responsible for the production and residence of immune cells. This study was performed to identify subpopulations of fish immune cells with different surface markers. Thus, Mabs against I.
제안 방법
- Hybridoma clones secreting antibodies were established by limiting-dilution method. The established hybridomas were stored in a liquid nitrogen tank and the supernatants were used fbr all experiments performed in this study.
- In order to investigate whether 7 different Mabs cross react with other kinds of fish immune cells, FACS analysis was performed with kidney and spleen cells from I. carp, common carp, catfish, tilapia, and rainbow trout. All antibodies positively responded with both I.
- In order to investigate whether 7 different Mabs cross react with other kinds of fish immune cells, FACS analysis was performed with kidney and spleen cells from I. carp, common carp, catfish, tilapia, and rainbow trout. All antibodies positively responded with both I.
- Figure 2 shows representative Wright's staining of cells (1, normal kidney mononuclear cells; 2, monocytes; 3, a mixed cell population of lymphocytes and monocytes; 4, lymphocytes). To study how many panned cells were surface Ig positive, FACScan analysis was performed with R I. cigs plus G RlgGFITC. Unexpectedly, all panned cell populations were surface Ig positive except for one separated by ICK 13-2 (figure 3).
- Figure 2 shows representative Wright's staining of cells (1, normal kidney mononuclear cells; 2, monocytes; 3, a mixed cell population of lymphocytes and monocytes; 4, lymphocytes). To study how many panned cells were surface Ig positive, FACScan analysis was performed with R I. cigs plus G RlgGFITC. Unexpectedly, all panned cell populations were surface Ig positive except for one separated by ICK 13-2 (figure 3).
- Thymidine incorporation assay was performed to identify how each purified cell population responded to three different kinds of mitogens, PHA, Con A and LPS (figure 4). Monocytes separated by ICK 17-4, 2-3 and 22-1 were efficiently reactive to Con A and PHA.
대상 데이터
- Israeli carp (I. carp), approximately 1 kg (±50 g), common carp, tilapia, catfish and rainbow trout were obtained from the aquaculture facility in the department of Marine Biomedical Science, Kunsan National University, Kunsan, Korea.
- Two female rabbits were injected with purified I. c a MIgG emulsified with Freund's complete adjuvant (FCA, Sigma) at 0.5 ml per rabbit.
- Two female rabbits were injected with purified I. c a MIgG emulsified with Freund's complete adjuvant (FCA, Sigma) at 0.5 ml per rabbit. Two weeks later, the rabbit was boosted with 0.
이론/모형
- The mononuclear cell pellet was resuspended in 5% BCS-DMEM, and then cell viability (>95%) was checked by tryphan blue exclusion method.
- carp mononuclear cells were collected at the interface between the DMEM and Histopaque-1077, and then washed twice with DMEM. The mononuclear cell pellet was resuspended in 5% BCS-DMEM, and then cell viability (>95%) was checked by tryphan blue exclusion method.
- carp kidney mononuclear cells. Hybridoma clones secreting antibodies were established by limiting-dilution method. The established hybridomas were stored in a liquid nitrogen tank and the supernatants were used fbr all experiments performed in this study.
- All steps were performed aseptically. The collected cells were used in Wright's staining and mitogen-induced proliferation assay. Further more, the panned cells were incubated with Rq I.
- Mabs-reacted I. carp kidney mononuclear cells were positively separated by panning method followed by Wright's stain. The stained cells were observed by optical microscopes.
후속연구
- The purified cell populations appeared as monocytes, lymphocytes or mixed cell population of monocytes and lymphocytes and they showed quite different proliferative patterns from mammalian cells. Further studies should be performed to identify the exact immune mechanisms occurring in teleost fishes. A better understanding of the fish immune system using the purified various kinds of immune-related cells will be helpful to design effective fish vaccines and monitor fish diseases in the course of therapy.
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