Fish and Shellfish Immunology 149 (2024) 109568

Contents lists available at ScienceDirect

Fish and Shellfish Immunology

journal homepage: www.elsevier.com/locate/fsi

Full length article

Establishment and characterization of a golden pompano (Trachinotus

blochii) fin cell line for applications in marine fish pathogen immunology
Jin-Feng Tong a, 1, Lang Yu a, 1, Rui-Hai Gan b, Li-Ping Shi a, Shao-Yang Bu a, Yue Gu a, Xin Wen a,
Jun-Long Sun a, Fei-Biao Song a, Li Zhou b, Jian-Fang Gui b, Jian Luo a, *
a
College of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), School of Marine Biology and Fisheries, Hainan Aquaculture Breeding
Engineering Research Center, Hainan Academician Team Innovation Center, Hainan University, Haikou, 570228, China
b
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, The Innovation Academy of Seed Design, Chinese Academy of Sciences,
Wuhan, 430072, China

A R T I C L E I N F O A B S T R A C T

Keywords: Pompano fishes have been widely farmed worldwide. As a representative commercial marine species of the
Golden pompano Carangidae family, the golden pompano (Trachinotus blochii) has gained significant popularity in China and
Fin cell line worldwide. However, because of rapid growth and high-density aquaculture, the golden pompano has become
Cytotoxic effect
seriously threatened by various diseases. Cell lines are the most cost-effective resource for in vitro studies and are
Poly I:C and LPS
Immune response
widely used for physiological and pathological research owing to their accessibility and convenience. In this
study, we established a novel immortal cell line, GPF (Golden pompano fin cells). GPF has been passaged over 69
generations for 10 months. The morphology, adhesion and extension processes of GPF were evaluated using light
and electron microscopy. GPF cells were passaged every 3 days with L-15 containing 20 % fetal bovine serum
(FBS) at 1:3. The optimum conditions for GPF growth were 28 ◦ C and a 20 % FBS concentration. DNA sequencing
of 18S rRNA and mitochondrial 16S rRNA confirmed that GPF was derived from the golden pompano. Chro­
mosomal analysis revealed that the number pattern of GPF was 48 chromosomes. Transfection experiments
demonstrated that GPF could be utilized to express foreign genes. Furthermore, heavy metals (Cd, Cu, and Fe)
exhibited dose-dependent cytotoxicity against GPF. After polyinosinic-polycytidylic acid (poly I:C) treatment,
transcription of the retinoic acid-inducible gene I-like receptor (RLR) pathway genes, including mda5, mita, tbk1,
irf3, and irf7 increased, inducing the expression of interferon (IFN) and anti-viral proteins in GPF cells. In
addition, lipopolysaccharide (LPS) stimulation up-regulated the expression of inflammation-related factors,
including myd88, irak1, nfκb, il1β, il6, and cxcl10 expression. To the best of our knowledge, this is the first study
on the immune response signaling pathways of the golden pompano using an established fin cell line. In this
study, we describe a preliminary investigation of the GPF cell line immune response to poly I:C and LPS, and
provide a more rapid and efficient experimental material for research on marine fish immunology.

1. Introduction golden pompano has become a highly valued marine fish with sub­
stantial commercial potential. In 2022, the domestic aquaculture output
The golden pompano (Trachinotus blochii), belonging to the Perci­ of golden pompano reached 245,400 tons, making it one of the top three
formes order, the Carangidae family, and the Pompano genus, is a varieties of mariculture fish in China [3]. However, with the expansion
commercially important marine species worldwide. In the early 2000s, of the breeding scale and increased breeding density, frequent outbreaks
there was a renewed interest in the golden pompano for aquaculture, of fish diseases have posed a challenge to the sustainable development of
and the Florida pompano (T. carolinus) in Southeastern U.S. coastal the golden pompano breeding industry. Several bacterial pathogens,
waters and the Indian pompano (T. mookalee) began to be commercially including Photobacterium damselae subsp. piscicida, Vibrio harveyi,
farmed [1,2]. Renowned for its delicious taste and rich nutrition, the Escherichia coli, and Edwardsiella tarda [4–7], have been reported to

* Corresponding author. Hainan University, Haikou, 570228, China.

E-mail address: [email protected] (J. Luo).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.fsi.2024.109568
Received 16 February 2024; Received in revised form 2 April 2024; Accepted 15 April 2024
Available online 16 April 2024
1050-4648/© 2024 Published by Elsevier Ltd.
J.-F. Tong et al. Fish and Shellfish Immunology 149 (2024) 109568

infect golden pompano and are the most common and harmful in its 2. Materials and methods
farming industry. Additionally, viral diseases, especially nerve necrosis
virus (NNV), are a common and severe threat to golden pompano 2.1. Primary cell culture and subculture
[8–10]. However, few studies have focused on the immunology of
golden pompano mainly because of a lack of suitable cell lines. There­ For the experiments, healthy golden pompano (T. blochii) weighing
fore, the establishment of appropriate cell lines is urgently needed to 80.0 ± 10.0 g were obtained from Hainan Blue Ocean Aquaculture Co.,
study disease control and breeding strategies for golden pompano. Ltd. in Lingshui, Hainan, China. These fish were kept in the laboratory in
Fish cell culture technology has emerged as a vital approach in ma­ tank (400 L each) filled with aerated seawater. Firstly, we soaked the
rine biology, playing a pivotal role in various fields. In addition to being fish in 1 % iodophor for 15 min anaesthetized them with 2–3 drops of
widely used in the generation of fish cells, including embryonic stem eugenol, and then wiped its surface with gauze soaked in 75 % ethanol.
cells, it has also been applied to fish virology, immunology, toxicology, The fin tissue was separated sterilely, and washed three times with
and other areas of research [11,12]. Among these, fish virology is one of Leibovitz’s L-15 medium (L-15, Thermo Fisher Scientifc Gibco,
the most widely explored fields in fish cell culture [13]. As a research Shanghai, CHN) containing 4 % antibiotic (400 IU/ml penicillin, 400
object, fish cells offer many advantages over living fish, including low μg/ml streptomycin; Gibco). Then, the fin tissue was cut into approxi­
cost, easy culture and preservation, good repeatability, and ease of mately 1-mm3 pieces and washed through a 100-mesh sieve. Finally, the
observation under a microscope. Cell lines derived from fish also com­ piece of fin tissue was seeded in a 25 cm2 tissue culture flask (Corning)
plement mammalian cell cultures in biomedical research, providing upside down in the incubator for 6 h, and then cultured in 5 ml complete
valuable insights into the pathophysiology and diagnostics of different medium [L-15 medium containing 20 % fetal bovine serum (FBS;
fish pathogens [14,15]. Fish cell lines are advantageous owing to their Gibco), 0.01 % Recombinant Human Basic Fibroblast Growth Factor
characteristics, such as growing in a wide temperature range, ease of (bFGF; PrimeGene), and 4 % antibiotics] at 28 ◦ C without CO2. The fresh
sampling, and higher homogeneity [16]. These cell lines are primarily medium would be gently replaced every 3 days. The primary cells were
used to study the pathogenesis of different fish infectious agents that grown to a confluence of 90%–95 % before being digested with 0.25 %
caused significant fish mortality and economic losses. Pathogenicity Tryptin EDTA solution (Gibco) for 2 min. The cells generally formed a
investigations also drive fish cell line development [17]. The immortal 100 % fused monolayer after 3 days and were cultured 1:3.
cell line described in this study was derived from golden pompano. Five
cell lines were derived from the snout, muscle, kidney, brain, and head
kidney of the golden pompano (https://www.cellosaurus.org/). These 2.2. Cell cryopreservation and recovery
cell lines have been used for Singapore grouper iridovirus (SGIV) and
red-spotted grouper nervous necrosis virus (RGNNV) isolation [18–22]. GPF cells were digested after forming a 100 % confluent cell
Despite the availability of many cell line resources, there still needs to be monolayer, and complete medium was added to terminate the digestion.
a breakthrough for direct and effective monitoring of contamination and GPF was centrifuged and resuspended in a cryopreservation medium
disease control in T. blochii. (10 % DMSO, 90 % FBS). Cryopreservation tubes were placed in – 20 ◦ C
Upon microbial invasion, cellular pattern recognition receptors for 1 h and kept at – 80 ◦ C for 12 h. GPF cells were moved to liquid
(PRRs) detect the corresponding pathogen associated molecular patterns nitrogen for long-term preservation. For cell recovery, GPF cells were
(PAMPs), initiating a cascade of signaling pathways to prevent disease extracted from liquid nitrogen and quickly thawed in 28 ◦ C water. The
[23]. As agonists of RLRs and the inflammatory signaling pathways, poly thawed cells were centrifuged and removed the supernatant. Then, GPF
I:C and LPS have been widely used in studies on immunity and inflam­ cells were resuspended in a complete medium and transformed into a 25
mation [24]. Poly I:C, a synthetic analogue of viral double-stranded cm2 tissue culture flask to add 5 ml complete medium. Cell adherence
RNA, induces innate antiviral responses by mimicking viral infection was observed in the following days.
through the activation of PRRs such as retinoic acid-inducible gene I
(RIG-I), laboratory of genetics and physiology 2, and melanoma
differentiation-associated gene 5 (MDA5) [25]. These receptors transmit 2.3. PCR amplification and sequencing
a signal to the mitochondrial antiviral-signaling protein (MAVS), which
activates the protein kinase TANK-binding kinase 1 (TBK1). TBK1 in­ To identify whether the established GPF cell line was derived from
teracts with the adapter protein mediator of IRF3 activation (MITA, also golden pompano, we amplified its 18S ribosomal RNA (rRNA) and
known as STING) localized in the mitochondria and endoplasmic re­ mitochondrial 16S rRNA gene as previously described [19,28]. The 6th
ticulum (ER), undergoing self-phosphorylation and subsequently phos­ generation of GPF cells was used to extract the genomic DNA by a Tissue
phorylating MITA. In this complex interaction, TBK1 further DNA kit (TIANGEN, Beijing, China). The 18S rRNA and mitochondrial
phosphorylates interferon regulatory factor 3 (IRF3) or IRF7, which are 16S rRNA fragments were amplified using primers (Table S1) according
transferred to the nucleus to induce the expression of IFN and other to the instruction of PrimeSTAR HS DNA polymerase (TaKaRa Bio,
antiviral effectors [26]. LPS, an endotoxin derived from gram-negative Shiga, Japan). Then, PCR products were determined by agarose gel and
bacteria such as E. coli, is frequently used in aquaculture research to sequenced. The acquired sequences were compared to known golden
stimulate the immune system [27]. It plays a similar role as an exoge­ pompano 18S rRNA and mitochondrial 16S rRNA sequences from NCBI
nous antigen and is used as an inflammatory inducer in some cases. LPS (GenBank Accession No. MK250487.1, EF613263.1).
can also be recognized by the immune system and stimulate the syn­
thesis and release of cytokines by signal transduction, inducing immune
stress. 2.4. Growth characteristics
In this study, we established an immortal cell line, GPF, derived from
the fin of the golden pompano. Under optimized culture conditions, GPF The optimum culture medium and serum concentration of GPF cells
showed better growth state and higher transfection efficiency. In addi­ were determined. GPF cells were inoculated into 6-well plates (Corning
tion, its susceptibility to heavy metals was also evaluated. Poly I: C was Inc.) at a concentration of 1 × 102 cells/well with three different me­
used to stimulate the innate antiviral responses of GPF, and LPS was diums (L-15, M199, and DMEM). Three different FBS concentrations
used to induce its immune system stimulation. We explored the GPF’s were 10 %, 15 %, and 20 %, respectively. GPF cells were collected by
different immune responses after stimulation with poly I:C and LPS. This trypsin digestion at the first, second, third, and fourth days of cell culture
is the first report on an established T. blochii fin cell line, providing novel and counted with hemocytometer. All experiments had three replicates
tools for investigating marine fish toxicology and immunology. and results were expressed as mean ± standard deviation (SD).

2
J.-F. Tong et al. Fish and Shellfish Immunology 149 (2024) 109568

2.5. Chromosome analysis 2.9. Poly I:C and LPS challenge

Chromosomes were analyzed in GPF cells at the 8th and 51st pas­ To explore the feasibility of GPF for immunological research, the
sages. A volume of 8 ml of cells (2 × 104 cell ml− 1) was seeded in 10-cm expression of GPF immune-related genes was evaluated after treatment
cell culture dishes (Corning Inc.) at 28 ◦ C for 24 h. Colchicine solution with poly I:C and LPS. Passage 55 GPF cells were seeded in a 6 well plate
was added into the cell culture dish with the final concentration of 1 μg/ and incubated with 20 % FBS medium for 24 h. When 90 % confluence,
ml. After incubation for 12 h, the cells were digested with trypsin and 1 μg/ml poly I:C or 10 μg/ml LPS was treated for 6, 12, 24, and 48 h
centrifuged at 350 g for 10 min. The cell precipitate was suspended in 8 respectively. Total RNA was isolated according to the instruction of
ml 0.075 M KCl solution at 37 ◦ C for 50 min at low osmotic temperature. reagent (Invitrogen). RNA quality was determined using agarose gel
Then, 2 ml pre-cooled Carnoy’s fixative (methanol to acetic acid = 3:1) electrophoresis. The spectrophotometer was used to assess the purity
was added into the cells for 5 min at room temperature. Centrifugation and yield of RNA (Nanodrop 2000, Thermo). The first-strand cDNA was
was performed again, and fixation was performed twice with 2 ml synthesized by using the GoScript Reverse Transcription System
fixative solution for 15 min. The fixed cells were dropped onto clean, (Promega) according to the manufacturer’s instructions. qPCR was
pre-cooled slides and stained with Giemsa solution for chromosomal performed with Fast SYBR Green master mix (BioRad) on a CFX96 Real-
counting under a Carl Zeiss upright fluorescence microscope (Axio Time System (BioRad). PCR conditions were as follows: 95 ◦ C for 5 min,
imager M2). then 40 cycles of 95 ◦ C for 20 s, 60 ◦ C for 20 s, 72 ◦ C for 20 s. The
expression of golden pompano β-actin was used as an internal control for
2.6. Cell cycle analysis normalization. β-actin and other primers of golden pompano inflam­
mation related genes and IFN related genes for qPCR analysis were
Cell cycle was analyzed in GPF cells at the 48th passage. GPF cells showed in Table S1. The specificity of the PCR amplification for all
were incubated in a 10-cm cell culture dish at 28 ◦ C for 24 h and reached primer sets was confirmed through dissociation curve analysis. The
to 70–80 % confluence. Then GPF cells were digested with trypsin and relative gene expression levels were calculated with 2-△△CT method. All
centrifuged at 350 g for 10 min. After washing GPF cells with PBS, the the samples were analyzed in triplicates.
supernatant was removed by centrifugation. And 1 ml DNA staining
solution (Multisciences, Beijing, CHN) was added to the cell precipita­ 2.10. Statistical analysis
tion to resuspend GPF cells by shocking for 10 s. Then GPF cells were
incubated at room temperature for 30 min in the dark. The fluorescence Statistical analysis was performed using a Student’s unpaired t-test,
signal was tested by CytoFLEX (Beckman), and the data were analyzed where a probability (p) value less than 0.05 was considered statistically
by FlowJo software. significant (*), and p < 0.01 was considered extremely significant (**).

2.7. Cytotoxicity assays of heavy metals in GPF cells 3. Results

Copper chloride (CuCl2), cadmium chloride (CdCl2), and ferric 3.1. Primary cell culture and subculture of GPF cells
chloride (FeCl3) were used to detect the toxicity of heavy metals to GPF
cells. GPF cells were seeded in a 96-well plate at 28 ◦ C for 24 h and Fin tissue blocks were affixed to a 25 cm2 tissue culture flask and
grown to 90 % confluence. Three kinds of heavy metals were diluted in placed in a 28 ◦ C incubator. After 7 days of culture, cells were observed
complete medium to the final concentration of 0.0001, 0.001, 0.01, 0.1, migrating from the fin tissue blocks (Fig. 1A). By day 16, the entire cell
1, and 10 mM, respectively, and then added into GPF cells. Cells of the layer covered 90 % of the flask bottom. After digestion of the primary
control group were cultured with the same amount of complete medium. cells using 0.25 % trypsin EDTA, they were divided into two flasks.
The cells were cultured in an incubator at 28 ◦ C for 24 h and then Within 3 days, the cells adhered and filled the flask bottom, forming the
replaced with fresh complete medium, the blank group had no cells but F1 generation (Fig. 1B). Subsequent subcultures occurred every 3 days
added the same amount of complete medium. The activity of GPF cells in a 1:3 ratio. The 20th generation cells were successfully revived after
was detected after incubation with CCK-8 (Servicebio, CHN) for 2 h. All freezing and some adhered to the flask 24 h after thawing. The adhesion
concentrations were set 3 parallel holes in 96-well plates and the results rate was up to 90 % after 48 h (Fig. 1C). To date, GPF cells have been
were representative of more than three independent experiments. subjected to more than 69 passages, with no observable changes in cell
size or morphology (Fig. 1D).
2.8. Cell transfection
3.2. Molecular identification of GPF cells
The transfection efficiency and gene expression of GPF cells were
measured by pEGFP-N3 plasmid expressing green fluorescent protein To authenticate the origin of GPF cells, the 18S rRNA and mito­
(GFP) to determine their suitability for studying viral pathogenesis and chondrial 16S rRNA genes were PCR-amplified from GPF cell DNA using
organism antiviral response. GPF cells at passages 50 were plated onto specific primers. The PCR products were approximately 455 bp and 418
coverslips in 6-well plates at a concentration of 2 × 104 cells/well and bp, respectively (Fig. S1). Sequence alignment analysis revealed 100 %
incubated at 28 ◦ C for 24 h. The GPF cells were transfected with 2 μg of similarity between the 18S rRNA and mitochondrial 16S rRNA se­
pEGFP-N3 plasmid using FishTrans Transfection Reagent (MeiSenTe quences of GPF cells and the known sequences of T. blochii, confirming
Biotechnology, CHN) and FuGENE HD Transfection Reagent (Promega). that GPF cells were derived from the golden pompano.
After transfection for 48 h, the cells were washed twice with PBS and
fixed with 4 % paraformaldehyde (PFA) for 1 h. The fixative was then 3.3. Growth characteristics
removed, and the cells were stained with 4′,6-diamidino-2-phenylindole
(DAPI) at a concentration of 1 μg/ml for 10 min in the dark at room To determine the optimum medium for GPF cells, three different
temperature. Subsequently, the coverslips were washed to remove mediums were tested. The results showed that GPF cells exhibited the
excess DAPI and placed onto glass slides. The stained cells on the cov­ fastest growth in L-15 medium (Fig. 2A). The cells thrived in L-15 me­
erslips were observed using a Carl Zeiss upright fluorescence microscope dium modified with 10–20 % FBS, with the optimum FBS concentration
(Axio imager M2) equipped with a 20x objective. The transfection effi­ being 20 % (Fig. 2B). The growth rate of GPF cultured with 15 % FBS
ciency was calculated by counting the number of GFP positive cells and was only slightly lower than that cultured with 20 % FBS. Considering
the total number of cells in 10 independent light fields. both growth characteristics and costs, a 15 % FBS concentration was

3
J.-F. Tong et al. Fish and Shellfish Immunology 149 (2024) 109568

Fig. 1. Establishment and subculture of GPF cells. Morphology of GPF monolayers at generation (A) 0th on Day 7, (B) 1st on Day 10. (C–D) The 20th and 50th
generations of GPF after cells resuscitation.

Fig. 2. Growth characteristics of GPF cells. Effects of different mediums (A) and FBS concentrations (B) on the growth of GPF cells. Results are presented as the mean
± SD of three independent experiments.

chosen for culturing GPF cells after 55 generations. 3.5. Cell cycle analysis

3.4. Chromosome analysis The GPF cell cycle was determined using DNA staining solution and
flow cytometry. The proliferating cell population was divided into the
The chromosome number was counted in GPF cells at the 8th and G0-G1 (DNA presynthetic phase, Blue), S (DNA synthesis phase, Green),
51st passages. Based on the results from 45 metaphase plates in GPF cells G2, and M -phases (mitosis, Red). Histograms showed two distinct peaks
at the 8th passage, the chromosome number ranged from 33 to 52 corresponding to the G0/G1 and G2/M phases (Fig. 4A). Cell debris was
(Fig. 3A). According to the modal distribution of chromosome number, excluded from the analysis, and the mitotic cell ratio was 4.26 %, the
the cell line displayed a chromosome number of 48 (Fig. 3B). Similarly, proportion of G0-G1 phase cells was 61.1 %, and the ratio of S phase
54 metaphase plates in GPF cells at the 51st passage were randomly cells was 26.0 % (Fig. 4B).
selected for chromosome analysis. The results also showed that the
number of chromosomes in GPF cells ranged from 33 to 52, and more
than one third of GPF cells had 48 chromosomes. Therefore, the modal 3.6. Cytotoxicity of heavy metals on GPF cells
chromosome number of GPF cell line was 48 (Fig. 3C and D). The
chromosome analysis of two generations of GPF cells showed that the In cytotoxicity tests with heavy metals, the viability of GPF cells
number of chromosomes was stable and did not change during cell gradually decreased with increasing concentrations of Cd, Cu, and Fe,
subculture. showing a dose-dependent effect (Fig. 5). The results indicated that Cd,
Cu, and Fe were toxic to GPF cells, with Cd showing the highest toxicity.
Under treatment with 0.01 mM Cd, 0.01 mM Cu, and 0.01 mM Fe, the
viability of GPF cells decreased significantly, and the survival rates
decreased to 79.70 %, 87.09 %, and 84.27 %, respectively. Furthermore,

4
J.-F. Tong et al. Fish and Shellfish Immunology 149 (2024) 109568

Fig. 3. Chromosome frequency distribution and morphological characteristics of GPF cells. Frequency distribution of chromosome numbers and metaphase in GPF
cells at the 8th (A–B) and 51st passages (C–D).

Fig. 4. Cell cycle of GPF cells. Peaks (A) and the percentage (B) of GPF cells at different stages of the cell cycle.

linear regression equations calculated in Excel revealed that the IC50 of effect on GPF cells, leading to fewer surviving cells compared to the
Cd was 0.01659 ± 0.0052 mM (Fig. 5A), which was significantly lower FishTrans transfection agent. These results suggest that a plasmid can be
than that of Cu (IC50 = 0.12302 ± 0.0308 mM) and Fe (IC50 = 0.23442 effectively transfected into GPF cells, making them suitable for in vitro
± 0.0587 mM) (Fig. 5B and C). foreign gene expression studies.

3.7. Transfection efficiency 3.8. Immune-related genes expression in GPF cells after poly I:C or LPS
challenge
After 48 h of transfection, a clear green fluorescence signal was
observed under a fluorescence microscope, indicating the successful To explore the feasibility of GPF as a tool for in vitro immunology
transfection of GPF cells with the pEGFP-N3 plasmid. Approximately 70 research, we used poly I:C and LPS to stimulate GPF cells, and significant
out of 220 cells in each field, showed GFP positivity when using Fish­ differences were observed in the expression levels of immune-related
Trans Transfection Reagent, resulting in transfection efficiency of genes. In the classical RLR pathway induced by poly I:C, the expres­
approximately 32 % for exogenous genes in GPF cells (Fig. 6A). In sion of upstream molecules of the RLR signaling pathway, including
addition, approximately 23 out of 90 cells in each field showed GFP mda5, mita, tbk1, irf3, and irf7, were up-regulated 4.5-, 5.9-, 4.1-, 5.3-,
positivity when using FuGENE HD Transfection Reagent (Fig. 6B). It is and 5.6- fold, respectively, after poly I:C treatment for 12 h (Fig. 7A–E).
worth noting that the FuGENE HD transfection agent had a damaging In addition, the expression of IFN-related genes, including ifn, viperin,

5
J.-F. Tong et al. Fish and Shellfish Immunology 149 (2024) 109568

Fig. 5. Cytotoxicity analysis of three heavy metals (Cd, Cu, and Fe) on GPF cells. (A) Viability of GPF cells in various concentrations of Cd solution. (B) Viability of
GPF cells in various concentrations of Cu solution. (C) Viability of GPF cells in various concentrations of Fe solution. Each bar presents mean ± SD of three inde­
pendent experiments. The asterisks indicate the significant differences (*p < 0.05, **p < 0.01).

Fig. 6. Expression of green fluorescent protein in GPF cells at the 50th passage. GFP fluorescence in pEGFP-N3-transfected GPF cells using FishTrans Transfection
Reagent (A) and FuGENE HD Transfection Reagent (B). Nucleus morphology of pEGFP-N3-transfected GPF cells stained by 6-diamidino-2-phenylindole (DAPI). The
yellow staining represents the merged images (original magnification, 20 × objective). Scale bars = 100 μm.

and isg15, were up-regulated 12-, 3162-, and 1256- fold, respectively However, challenges arise from intensive farming practices and eutro­
(Fig. 7F–H). In the LPS-stimulated classical pathway, the expression of phic seawater conditions, leading to severe disease issues. At present,
the myeloid differentiation factor 88 (myd88), il-1r–associated kinase 1 fish cell lines are essential research tools and have broad applications in
(irak1), nuclear factor kappa b (nfκb), cxc chemokine ligand 10 (cxcl10), marine biology. In this study, we established and characterized a fin cell
interleukin 1β (il1β), and il6 genes significantly increased after treatment line of T. blochii. PCR analysis of 18S rRNA and mitochondrial 16S rRNA
with LPS for 24 h and 48 h, and being up-regulated 2.7-, 12.3-, 3.6-, genes confirmed the origin of GPF cells as T. blochii (Fig. S1). 18S rRNA
39.4-, 46.5-, and 29.1- fold, respectively, after 24 h of LPS treatment and mitochondrial 16S rRNA have highly and moderately conserved
(Fig. 8A–F). These results indicate that the expression of immune-related sequence regions as well as highly varied sequence regions, which may
genes changes when GPF cells are stimulated by poly I:C or LPS, which reflect differences between genera. Additionally, the present study
can be used for immune research in vitro. demonstrated that GPF cells are suitable for foreign gene expression,
pathological studies, and in vitro cytotoxicity assessments.
4. Discussion The time for cells to migrate from fin explants varied, resulting in
diverse cell types and morphologies. After culturing in 20 % FBS L-15
The golden pompano (T. blochii) is gaining popularity among Chinese medium, the cells gradually assumed a fibroblast-like morphology
consumers owing to its delicate meat quality and high nutritional value. (Fig. 1). Similar to other eukaryotic cell cultures, fish cell cultures

6
J.-F. Tong et al. Fish and Shellfish Immunology 149 (2024) 109568

Fig. 7. Effects of poly I:C on expression of immune-related genes in GPF cells. Total RNAs of GPF cells treated with poly I:C were extracted to examine the transcript
levels of mda5, mita, tbk1, irf3, irf7, ifn, viperin, and isg15 (A–H). The β-actin of golden pompano was used as an internal control for normalization and the relative
expression is represented as fold induction relative to the expression level in control cells. Error bar represents SD obtained by measuring each sample in triplicate.
Asterisks indicate significant differences from control (*p < 0.05, **p < 0.01).

require a growth medium providing nutrients and growth factors [29]. cytometry analysis revealed that the G1-G2 phase cell number was more
In this study, the growth rate of GPF cells was the fastest in L-15 medium than twice that of the S phase, determining the cell replication/division
without additional CO2 buffering, consistent with its common use in cycle (Fig. 4). This suggested that the GPF cell line had good prolifera­
marine fish cell lines, such as Cromileptes altivelis kidney (CAK) cells [30] tion ability and could be passaged continuously. Successful and efficient
and Epinephelus moara brain (EMB) and kidney (EMK) cells [31,32]. L-15 transfection of foreign genes is one of the critical uses of cell lines, which
medium is a balanced salt solution, with a high concentration of amino is the basis for studying the function of foreign genes in cell lines. Our
acids that provides a more effective buffering function and improves cell study showed that exogenous GFP could be expressed efficiently in GPF
reproduction and growth by using galactose as a carbon source, avoiding cells, and the expression efficiency was approximately 32 % (Fig. 6).
the reliance on high levels of CO2 as a buffering system. Therefore, the GPF had a little better transfection efficiency for foreign genes,
effective buffer system of L-15 medium can prevent excessive fluctua­ compared with other marine fish cells. For example, the Japanese
tion of pH and ensure long-term cell culture, which is consistent with the flounder brain cell line has a transfection efficiency of 30 %, the trans­
growth characteristics of fish cells [30]. In addition, FBS is the most fection efficiency of E. coioides intestine cell line is 18.7 %, and the
commonly used culture medium supplement. For most fish cell cultures, transfection efficiency is only 3 % in the Larimichthys crocea embryo cell
10–20 % FBS are ideal for growth. For example, considering the cost and line [34,36,37]. Moreover, the GPF cell line could be used for studying
growth of cells, the best concentration of FBS is 10 % for the Epinephelus the function of foreign genes. Similarly, foreign GFP genes can also be
lanceolatus head kidney cell line and 15 % for the Epinephelus coioides gut expressed in other fish fin cell lines, such as the Pangasianodon hypo­
cell line [33,34]. Here, the optimum concentration of FBS for GPF cell phthalmus fin cell [38], the Pterophyllum scalare fin cell, and the Mega­
culture was determined to be 20 % (Fig. 2). lobrama amblycephala fin cell [39,40].
Chromosomes are involved in the mechanism of inheritance and the Fish cell lines are valuable alternatives for testing the toxicology of
expression of the genetic characteristics through successive generations heavy metals [12]. Many new cell lines have been developed in the last
[35]. With the increase in the number of cell passages, chromosome decade to study the effects of heavy metals [21,41]. Recently, Cyprinus
variation will gradually increase. Chromosomal analysis at the 8th and carpio haematopterus muscle cells (CCM) were treated with heavy metals
51st passages revealed a stable modal chromosome number of 48, (Hg, Cu, and Cd), and the cytotoxicity of heavy metals in CCM cells
indicating chromosomal stability during cell subculture (Fig. 3). Flow occurred in a dose-dependent manner [42]. Wei et al. reported that fish

7
J.-F. Tong et al. Fish and Shellfish Immunology 149 (2024) 109568

Fig. 8. Effects of LPS on expression of immune-related genes in GPF cells. Total RNAs of GPF cells treated with LPS were extracted to examine the transcript levels of
myd88, irak1, nfκb, cxcl10, il1β, and il6 (A–F). The β-actin of golden pompano was used as an internal control for normalization and the relative expression is
represented as fold induction relative to the expression level in control cells. Error bar represents SD obtained by measuring each sample in triplicate. Asterisks
indicate significant differences from control (*p < 0.05, **p < 0.01).

cell lines from different species might have different sensitivities to and characterized. GPF had better transfection efficiency for foreign
heavy metals: Hg, Cu, and Cd were all toxic to Cromileptes altivelis kidney genes and was susceptible to heavy metals (Cd, Cu, and Fe). The immune
cells (CAK), and Hg had the strongest cytotoxicity to CAK cells [30]. In response of GPF to poly I:C and LPS stimulation was explored, and GPF
this study, Cd, Cu, and Fe were toxic to GPF cells, and Cd was the most provided the experimental materials for an in vitro study of golden
toxic (Fig. 5), which could be used for subsequent cytotoxicity studies. pompano toxicology and immunology. This research will facilitate other
Similarly, among the four heavy metals Cr, Cd, Zn, and Cu, Cd was the immune-related studies of pompano fishes, and provide guidance for the
most toxic to channel catfish ovary cells [43]. It’s worth noting that the establishment of other pompano fish cell lines.
IC50 of Cd for GPF cells was 0.01659 ± 0.0052 mM, which was lower
than the IC50 of Cd (0.0274 ± 0.0106 mM) for CAK cells and the IC50 of Funding
Cd (0.01819 mM) for CCM cells. These results indicate that the GPF cell
line is more sensitive to Cd and can thus be used to detect low concen­ This work was supported by the National Key R&D Program of China
trations of Cd in water environments. (2022YFD2400504), the project of Hainan Yazhou Bay Seed Laboratory
Cell lines have contributed significantly to in vitro pathological (B21HJ0105), the project of Hainan Provincial Postdoctoral Science
studies, especially in fish virology [44]. Non-specific fish defenses can be Foundation, and Hainan Provincial Natural Science Foundation of China
activated by natural or synthetic immune stimulants, including bacterial (324QN199).
LPS and poly I:C, which mimics viral double-stranded RNA [45–47].
Poly I:C, a viral mimic, stimulates the organism to produce an immune Availability of data and materials
response through the RLR pathway [48]. In this study, poly I:C stimu­
lation significantly increased the expression of mda5 in GPF cells, and All data generated and analyzed during this study are included in this
the expression of mita, tbk1, irf3, and irf7 increased correspondingly. In published article.
addition, IFN-related genes, including ifn, viperin, and isg15, were
remarkably up-regulated (Fig. 7). Therefore, poly I:C was recognized by Conflicts of interest
MDA5 and induced the IFN response through the RLR pathway in GPF
cells. LPS is highly conserved in gram-negative bacteria and can effec­ The authors declare no conflict interest.
tively induce inflammatory responses, regulate the immune system, and
affect disease susceptibility [27]. After TLR4 recognizes LPS in an Consent for publication
extracellular environment, it triggers the MyD88-dependent pathway
mediated by MyD88 to promote the synthesis of cytokines and chemo­ Not applicable.
kines [49]. In this study, after LPS stimulation, expression of the
inflammatory-related factors, including myd88, irak1, nfκb, il1β, il6, and Ethics approval and consent to participate
cxcl10 was up-regulated (Fig. 8). Similarly, LPS affects the
MyD88-IRAKs-NFκB pathway in CCM cells, induces the expression of All experiments were performed according to the Guidelines for the
il1β, il8, and il10, and enhances a specific immune response [42]. Care and Use of Laboratory Animals in China. All experimental pro­
Whether LPS induces another myd88-independent pathway mediated by cedures and sample collection were approved by the Institutional Ani­
interferon-TIR domain adaptor protein (TRIF) remains to be determined. mal Care and Use Committee (IACUC) of the College of Ocean of Hainan
In conclusion, golden pompano fin cell line (GPF) was established University, Hainan, China.

8
J.-F. Tong et al. Fish and Shellfish Immunology 149 (2024) 109568

CRediT authorship contribution statement [14] J. Lu, D. Xu, L. Lu, A novel cell line established from caudal fin tissue of Carassius
auratus gibelio is susceptible to cyprinid herpesvirus 2 infection with the induction
of apoptosis, Virus Res. 258 (2018) 19–27.
Jin-Feng Tong: Investigation, Visualization, Writing – original draft. [15] M. Imajoh, H. Fujioka, K. Furusawa, K. Tamura, K. Yamasaki, S. Kurihara,
Lang Yu: Investigation, Visualization, Writing – original draft. Rui-Hai J. Yamane, K. Kawai, S. Oshima, Establishment of a new cell line susceptible to
Gan: Methodology, Visualization. Li-Ping Shi: Methodology, Investi­ Cyprinid herpesvirus 3 (CyHV-3) and possible latency of CyHV-3 by temperature
shift in the cells, J. Fish. Dis. 38 (6) (2015) 507–514.
gation. Shao-Yang Bu: Methodology, Investigation. Yue Gu: Method­ [16] N.C. Bols, P.H. Pham, V.R. Dayeh, L.E.J. Lee, Invitromatics, invitrome, and
ology, Investigation. Xin Wen: Visualization. Jun-Long Sun: invitroomics: introduction of three new terms for in vitro biology and illustration
Visualization. Fei-Biao Song: Visualization. Li Zhou: Writing – review of their use with the cell lines from rainbow trout, In Vitro Cell. Dev. Biol. Anim. 53
(5) (2017) 383–405.
& editing. Jian-Fang Gui: Writing – review & editing. Jian Luo: [17] N.S. Nagpure, A.K. Mishra, A.S. Ninawe, A. Rasal, A. Dubey, A. Kumar,
Conceptualization, Writing – review & editing. M. Goswami, R. Kumar, J.K. Jena, Molecular and cytogenetic characterization of
fish cell lines and its application in aquatic research, Natl. Acad. Sci. Lett. 39 (1)
(2016) 11–16.
Declaration of competing interest [18] P. Li, L. Zhou, S. Ni, M. Xu, Y. Yu, J. Cai, S. Wei, Q. Qin, Establishment and
characterization of a novel cell line from the brain of golden pompano (Trachinotus
ovatus), In Vitro Cell. Dev. Biol. Anim. 52 (4) (2016) 410–418.
The authors declare no conflict of interest. [19] Y. Yu, S. Wei, Z. Wang, X. Huang, Y. Huang, J. Cai, C. Li, Q. Qin, Establishment of a
new cell line from the snout tissue of golden pompano Trachinotus ovatus, and its
application in virus susceptibility, J. Fish. Biol. 88 (6) (2016) 2251–2262.
Data availability
[20] P. Li, L. Zhou, S. Wei, M. Yang, S. Ni, Y. Yu, J. Cai, Q. Qin, Establishment and
characterization of a cell line from the head kidney of golden pompano Trachinotus
No data was used for the research described in the article. ovatus and its application in toxicology and virus susceptibility, J. Fish. Biol. 90 (5)
(2017) 1944–1959.
[21] L. Zhou, P. Li, J. Liu, S. Ni, Y. Yu, M. Yang, S. Wei, Q. Qin, Establishment and
Acknowledgments Characterization of a Mid-kidney Cell Line Derived from Golden Pompano
Trachinotus Ovatus, a New Cell Model for Virus Pathogenesis and Toxicology
Studies, 2017, pp. 320–327.
The authors were grateful to all the laboratory members for contin­
[22] L. Zhang, K. Zhu, H. Guo, B. Liu, B. Liu, N. Zhang, D. Zhang, Establishment and
uous technical advice and helpful discussion. characterization of a muscle cell line from golden pompano (Trachinotus ovatus)
and its application in viral susceptibility, Aquaculture 580 (2024) 740286.
[23] S.W. Brubaker, K.S. Bonham, I. Zanoni, J.C. Kagan, Innate immune pattern
Appendix A. Supplementary data recognition: a cell biological perspective, Annu. Rev. Immunol. 33 (2015)
257–290.
Supplementary data to this article can be found online at https://doi. [24] B. Robertsen, Modulation of the non-specific defence of fish by structurally
conserved microbial polymers, Fish Shellfish Immunol. 9 (4) (1999) 269–290.
org/10.1016/j.fsi.2024.109568.
[25] T. Kawai, K. Takahashi, S. Sato, C. Coban, H. Kumar, H. Kato, K.J. Ishii,
O. Takeuchi, S. Akira, IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type
References I interferon induction, Nat. Immunol. 6 (10) (2005) 981–988.
[26] S.N. Chen, P.F. Zou, P. Nie, Retinoic acid-inducible gene I (RIG-I)-like receptors
(RLRs) in fish: current knowledge and future perspectives, Immunology 151 (1)
[1] C.R. Weirich, K.L. Riley, M. Riche, K.L. Main, P.S. Wills, G. Illán, D.S. Cerino, T.
(2017) 16–25.
J. Pfeiffer, The status of Florida pompano, Trachinotus carolinus, as a commercially
[27] L. Mazgaeen, P. Gurung, Recent advances in lipopolysaccharide recognition
ready species for U.S. Marine aquaculture, J. World Aquacult. Soc. 52 (3) (2021)
systems, Int. J. Mol. Sci. 21 (2) (2020) 379.
731–763.
[28] L. Wang, Z. Cao, Y. Liu, Y. Xiang, Y. Sun, Y. Zhou, S. Wang, W. Guo, Establishment
[2] M. Sekar, R. Ranjan, B. Xavier, S. Ghosh, V. Pankyamma, B. Ignatius, I. Joseph,
and characterization of a new cell line from the muscle of humpback grouper
G. Achamveetil, Species validation, growth, reproduction and nutritional
(Cromileptes altivelis), Fish Physiol. Biochem. 46 (6) (2020) 1897–1907.
perspective of Indian pompano, Trachinotus mookalee–a candidate species for
[29] C. Zhong, Y. Tao, M. Liu, X. Wu, Y. Yang, T. Wang, Z. Meng, H. Xu, X. Liu,
diversification in coastal mariculture, Aquaculture 545 (2021) 737212.
Establishment of a spermatogonial stem cell line with potential of meiosis in a
[3] Bureau of Fisheries, Ministry of Agriculture and Rural Affairs of the People’s
hermaphroditic fish, Epinephelus coioides, Cells 11 (18) (2022) 2868.
republic of china. China Fishery Statistical Yearbook, China Agriculture Press,
[30] C. Wei, X. Yang, M. Kang, Z. Cao, Y. Sun, Y. Zhou, An established kidney cell line
Beijing, 2023.
from humpback grouper (Cromileptes altivelis) and its susceptibility to bacteria and
[4] B. Sun, Y. Lei, Z. Cao, Y. Zhou, Y. Sun, Y. Wu, S. Wang, W. Guo, C. Liu, TroCCL4, a
heavy metals, Fish Physiol. Biochem. 48 (3) (2022) 521–533.
CC chemokine of Trachinotus ovatus, is involved in the antimicrobial immune
[31] X. Liu, Y. Wu, S. Wei, N. Wang, P. Li, Y. Li, N. Zhang, Q. Qin, S. Chen,
response, Fish Shellfish Immunol. 86 (2019) 525–535.
Establishment and characterization of a kidney cell line from kelp grouper
[5] Y. Zhou, X. Chen, Z. Cao, J. Li, H. Long, Y. Wu, Z. Zhang, Y. Sun, R848 is involved
Epinephelus moara, Fish Physiol. Biochem. 44 (1) (2018) 87–93.
in the antibacterial immune response of golden pompano (Trachinotus ovatus)
[32] X.F. Liu, Y.H. Wu, S.N. Wei, N. Wang, Y.Z. Li, N.W. Zhang, P.F. Li, Q.W. Qin, S.
through TLR7/8-MyD88-NF-κB-Signaling pathway, Front. Immunol. 11 (2021)
L. Chen, Establishment and characterization of a brain-cell line from kelp grouper
617522.
Epinephelus moara, J. Fish. Biol. 92 (2) (2018) 298–307.
[6] R. Wang, J. Feng, Y. Su, L. Ye, J. Wang, Studies on the isolation of Photobacterium
[33] Z. Liu, Z. Xin, Z. Ya, Q. Qiwei, H. Xiaohong, H. Youhua, Establishment of a cell line
damselae subsp. Piscicida from diseased golden pompano (Trachinotus ovatus
from the head kidney of giant grouper (Epinephelus lanceolatus) and its
Linnaeus) and antibacterial agents sensitivity, Vet. Microbiol. 162 (2–4) (2013)
susceptibility to fish viruses, Aquaculture Reports 21 (6) (2021) 100899.
957–963.
[34] J. Fan, J. Zheng, W. Lai, S. Liu, X. Liang, Q. Wang, Y. Huang, J. Wei, X. Huang,
[7] C. Zhou, X. Ge, H. Lin, J. Niu, Effect of dietary carbohydrate on non-specific
Q. Qin, S. Zhou, Establishment of a new intestinal cell line from the grouper
immune response, hepatic antioxidative abilities and disease resistance of juvenile
(Epinephelus coioides) for aquatic virus pathogenesis, Aquaculture 562 (2023)
golden pompano (Trachinotus ovatus), Fish Shellfish Immunol. 41 (2) (2014)
738771.
183–190.
[35] H. Jing, X. Lin, L. Xu, L. Gao, M. Zhang, N. Wang, S. Wu, Establishment and
[8] H. Ma, W. Wen, Y. Su, J. Feng, L. Xu, C. Peng, Z. Guo, Epidemiological
characterization of a heart-derived cell line from goldfish (Carassius auratus), Fish
characterization of VNNV in hatchery-reared and wild marine fish on Hainan
Physiol. Biochem. 43 (4) (2017) 977–986.
Island, China, and experimental infection of golden pompano (Trachinotus ovatus)
[36] Z. Sun, Y. Ren, Y. Zhang, Y. Yang, G. Wang, Z. He, Y. Liu, W. Cao, Y. Wang, Y. Fu,
juveniles, Arch. Virol. 160 (12) (2015) 2979–2989.
J. Hou, Establishment and characterization of a cell line from the brain of the
[9] Y. Su, H. Xu, H. Ma, J. Feng, W. Wen, Z. Guo, Dynamic distribution and tissue
Japanese flounder (Paralichthys olivaceus) and its application in the study of viral
tropism of nervous necrosis virus in juvenile pompano (Trachinotus ovatus) during
infection, Aquaculture 562 (2023) 738825.
early stages of infection, Aquaculture 440 (2015) 25–31.
[37] Z. Zheng, H. Chi, X. Liu, X. Yang, X. Chen, Y. Pan, H. Gong, A new embryonic cell
[10] H. Xu, J. Feng, Z. Guo, Y. Ou, J. Wang, Detection of red-spotted grouper nervous
line YCE1 from large yellow croaker (Larimichthys crocea) and its susceptibility to
necrosis virus by loop-mediated isothermal amplification, J. Virol Methods 163 (1)
large yellow croaker iridovirus, Aquaculture 565 (2023) 739079.
(2010) 123–128.
[38] P. Soni, P.K. Pradhan, T.R. Swaminathan, N. Sood, Development, Characterization
[11] L.E. Hightower, J.L. Renfro, Recent applications of fish cell culture to biomedical
and Application of a New Epithelial Cell Line from Caudal Fin of Pangasianodon
research, J. Exp. Zool. 248 (3) (1988) 290–302.
Hypophthalmus (Sauvage 1878), vol. 182, 2018.
[12] R.S. Thangaraj, L. Narendrakumar, P. Prasannan Geetha, A.R. Shanmuganathan,
[39] T.R. Swaminathan, R. Kumar, P.M. Jency, R. Charan, M.U. Syamkrishnan, V.
A. Dharmaratnam, S.R. Nithianantham, Comprehensive update on inventory of
S. Basheer, N. Sood, J.K. Jena, A new fish cell line derived from the caudal fin of
finfish cell lines developed during the last decade (2010–2020), Rev. Aquacult. 13
freshwater angelfish Pterophyllum scalare: development and characterization,
(4) (2021) 2248–2288.
J. Fish. Biol. 89 (3) (2016) 1769–1781.
[13] X. Huang, Y. Huang, Z. Ouyang, Q. Qin, Establishment of a cell line from the brain
of grouper (Epinephelus akaara) for cytotoxicity testing and virus pathogenesis,
Aquaculture 311 (1–4) (2011) 65–73.

9
J.-F. Tong et al. Fish and Shellfish Immunology 149 (2024) 109568

[40] D.M. Zhu, K. Yang, W.M. Wang, W. Song, Establishment and characterization of a [45] C. Guo, J.Z. Ye, M. Song, X.X. Peng, H. Li, Poly I:C promotes malate to enhance
fin cell line from blunt snout bream, Megalobrama amblycephala, Fish Physiol. innate immune response against bacterial infection, Fish Shellfish Immunol. 131
Biochem. 39 (6) (2013) 1399–1410. (2022) 172–180.
[41] P. Morcillo, E. Chaves-Pozo, J. Meseguer, M.A. Esteban, A. Cuesta, Establishment [46] J. Saravia, K. Paschke, J.P. Pontigo, D. Nualart, J.M. Navarro, L. Vargas-Chacoff,
of a new teleost brain cell line (DLB-1) from the European sea bass and its use to Effects of temperature on the innate immune response on Antarctic and sub-
study metal toxicology, Toxicol. Vitro 38 (2017) 91–100. Antarctic fish Harpagifer antarcticus and Harpagifer bispinis challenged with two
[42] S. Zhi, J. Wang, Y. Wang, L. Yang, C. Qin, X. Yan, M. Zhao, M. Liu, L. Qu, G. Nie, immunostimulants, LPS and Poly I:C: In vivo and in vitro approach, Fish Shellfish
Establishment and characterization of Yellow River carp (Cyprinus carpio Immunol. 130 (2022) 391–408.
haematopterus) muscle cell line and its application to fish virology and [47] A. Martinez-Lopez, S.D. Tyrkalska, F. Alcaraz-Perez, I. Cabas, S. Candel, M.
immunology, Fish Shellfish Immunol. 139 (2023) 108859. F. Martinez, M.P. Sepulcre, D. Garcia-Moreno, M.L. Cayuela, V. Mulero, Evolution
[43] F. Tan, M. Wang, W. Wang, Y. Lu, Comparative evaluation of the cytotoxicity of LPS recognition and signaling: the bony fish perspective, Dev. Comp. Immunol.
sensitivity of six fish cell lines to four heavy metals in vitro, Toxicol. Vitro 22 (1) 145 (2023) 104710.
(2008) 164–170. [48] J. He, S. Hu, Y. Xie, Y. Wei, Q. Zhang, X. Pi, Z. Qi, Molecular characterization and
[44] C. Fierro-Castro, L. Barrioluengo, P. Lopez-Fierro, B.E. Razquin, A.J. Villena, Fish expression analysis of TRIF, TRAF6, and TBK1 of golden pompano (Trachinotus
cell cultures as in vitro models of inflammatory responses elicited by ovatus), Fish Shellfish Immunol. 127 (2022) 604–610.
immunostimulants. Expression of regulatory genes of the innate immune response, [49] A. Ciesielska, M. Matyjek, K. Kwiatkowska, TLR4 and CD14 trafficking and its
Fish Shellfish Immunol. 35 (3) (2013) 979–987. influence on LPS-induced pro-inflammatory signaling, Cell. Mol. Life Sci. 78 (4)
(2021) 1233–1261.

10