Trypan

The above feeding study provided preliminary data on the in vivo nutritional regulation of elovl4 and elovl5. To confirm and support these data, an in vitro study was conducted to further investigate the role of certain transcription factors on the regulation of elovl4 and elovl5 elongases in L. crocea. Hepatocytes were isolated from five yellow croaker individuals (~50 g) starved for 24 h according to the published protocols^{49 (link)} with slight modification^{50 (link), 51 (link)}. Briefly, croaker were anesthetized with MS 222 and the branchial arch cut followed by immersion in 70% ethanol for 3 min to sterilize the external surface. Liver tissues were excised aseptically and rinsed twice with phosphate buffered saline (PBS, pH 7.4 at 4 °C) supplemented with amphotericin-B (25 μg mL⁻¹), streptomycin (100 μg mL⁻¹) and penicillin (100 IU mL⁻¹). Thereafter, the liver was aseptically minced into 1 mm³ pieces, followed by digestion in 0.25% sterile trypsin at room temperature for 15 min. Trypsin was neutralized with Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) containing 20% fetal bovine plasma (FBS, Invitrogen, USA). The cell suspension was collected and filtered through sterile 75 μm mesh, followed by centrifugation at low speed (100 g, 5 min) and the supernatant discarded. Isolated cells were washed in red blood cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) for 2 min at 4 °C. Cell viability was evaluated using a hemocytometer under an inverted microscope after the cells were stained with 0.4% Trypan Blue. The hepatocytes were re-suspended in DMEM medium containing 1 mM glutamine, 20% FBS, penicillin (100 IU mL⁻¹) and streptomycin (100 μg mL⁻¹). Cells suspensions with more than 95% cell viability were used for the subsequent experiments.
Croaker primary hepatocyte suspensions were incubated with fatty acid (DHA or EPA) to confirm the influence of n-3 LC-PUFA on the transcription of the elongases (elovl4 and elovl5) and transcription factors (lxrα and srebp-1) in vivo. Fatty acid (DHA and EPA, Cayman Chemical Co., USA) was supplemented to cells in the form of BSA/fatty acid complexes that were prepared at 10 mM concentration according to Ou et al.^{52 (link)} and stored at −20 °C. Additionally, inhibitors/agonists of transcription factors were used to clarify the role of the transcription factors on the regulation of elovl4 and elovl5 elongases in L. crocea. GW3965 HCl (Selleckchem, Shanghai, China) was used as an LXRα agonist whereas FGH10019 (MCE, USA) was used as a SREBP-1 inhibitor, respectively. Cells were seeded in 6-well plates with a density of 2 × 10⁶ viable cells per well in DMEM/F12 (Gibco) containing 20% FBS, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin, followed by incubation for 24 h. The hepatocytes were then washed and incubated for 1 h in FBS-free DMEM/F12 medium prior to incubation with EPA, DHA and the above inhibitors or agonists in triplicate wells. After incubation, cells were lysed in the wells and harvested for RNA extraction.

Fresh samples from biopsy or surgery were isolated and transported rapidly to the research facility. Tissues were transported in a sterile culture dish with 10 ml 1× Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher, Cat. no. 14190144) on ice (4 °C) to remove the residual tissue storage solution, then minced into 1–3 mm³ pieces in another culture dish. We used 10 mg type I collagenase (Sigma, Cat. no. C0130) dissolved in 10 ml RPMI 1640 medium (Thermo Fisher, Cat. no. 10-040-CM) with 10% fetal bovine serum (FBS; Thermo Fisher, Cat. no. SV30087.02) to digest the tissues. Tissues were dissociated at 37 °C with a shaking speed of 50 r.p.m. Cell suspensions were filtered using a 70-um nylon cell strainer and red blood cells were removed by 1× Red Blood Cell Lysis Solution (Thermo Fisher, Cat. no. 00-4333-57). Dissociated cells were washed with 1× DPBS containing 2% FBS. Cells were stained with 0.4% Trypan blue (Thermo Fisher, Cat. no. 14190144) to check the viability on Countess® II Automated Cell Counter (Thermo Fisher).

For cell growth analysis, a 2 ml aliquot of LCL wash plated at 5 × 10⁴ cells/ml per well in 12‐well plates and JRS cells were plated at 5 × 10⁴ cells per well in 6‐well plates. Following 0–96 h incubations in medium with or without Asn (RPMI for LCL, DMEM for JRS cells), cells were collected to determine viable cell numbers using the Applied Biosystems Countess II FL automated cell counter per the manufacturer's trypan blue staining protocol. All reported values are trypan‐excluding, viable cells only.