Neonatal mice were administered Ad-AHAL2 at a dose of 5.9 × 1011 IFU/kg. Blood samples were collected on the indicated day points and placed on ice for 1 h. The serum was collected following centrifugation at 7000 g at 4°C for 15 min. The serum alanine aminotransferase (ALT) and aspartateaminotransferase (AST) levels in the serum were determined using a transaminase-CII kit (Wako Pure Chemical). For the histopathological examination of liver sections, the livers were recovered from mice 2 days after Ad vector administration. The livers were washed, fixed in 10% buffered formalin (Wako Pure Chemical), embedded in paraffin, and processed for histology. For evaluation of Ad vector-mediated cardiac toxicity, the serum creatine kinase (CK) levels were determined using a Creatine Kinase Assay kit (BioAssay Systems, Hayward, CA).
Transaminase cii kit
Manufactured by Fujifilm
Sourced in Japan
The Transaminase CII kit is a laboratory diagnostic tool used for the quantitative determination of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activities in biological samples. The kit provides the necessary reagents and protocols to measure these enzymes, which are commonly used as markers of liver function and tissue damage.
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Lab products found in correlation
Buffered formalin, by Fujifilm (2 mentions)
Lunar piximus 2, by GE Healthcare (1 mentions)
Rat insulin ria kit, by Merck Group (1 mentions)
Thunderbird sybr qpcr mix, by Toyobo (1 mentions)
Accu chek glucometer, by Roche (1 mentions)
Cholesterol e, by Fujifilm (1 mentions)
Free cholesterol e, by Fujifilm (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Wild type cd1 mice, by Charles River Laboratories (1 mentions)
Rat albumin elisa quantitation set, by Fortis Life Sciences (1 mentions)
8 protocols using transaminase cii kit
1
Assessing Adenoviral Vector Toxicity
2
Metabolic Phenotyping in Mice
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Mouse body weights were monitored weekly and body composition was assessed by dual-energy X-ray absorptiometry (DEXA; Lunar PIXImus2, GE Healthcare). Insulin and glucose tolerance tests were performed as described previously (Loh et al., 2009 (link)). Blood was collected from the retro-orbital sinus and fed (9 am) and fasted (12 h overnight fast) plasma insulin levels determined using a Rat insulin RIA kit (Merck Millipore, CA) and the corresponding blood glucose levels determined with an Accu-Check glucometer. Fed serum ALT and AST levels were determined using the Transaminase CII kit from Wako Pure Chemical Industries (Osaka, Japan) as described previously (Wiede et al., 2011 (link)) and serum CXCL9 (CXCL9 Mouse ELISA Kit, Thermo Fisher Scientific, Waltham, WA), FGL1 (USCN Life Science, Wuhan, China) and LCN2 (NGAL Mouse ELISA Kit, Thermo Fisher Scientific, Waltham, WA) levels measured by ELISA according to the manufacturers’ instructions.
3
Adenoviral Vector Liver Toxicity Assessment
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Ad vectors were intravenously administered into C57BL/6 and Rag2/Il2rγ double-knockout mice at a dose of 1 × 1010 IFU/mouse. The blood samples were collected via retro-orbital bleeding at the indicated days, and the serum samples were obtained by centrifugation. The serum ALT levels were determined using a transaminase-CII kit (Wako Pure Chemical Industries). The serum levels of ALP, LDH, and LAP were analyzed at the Oriental Yeast Corporation (Tokyo, Japan). For the histopathological examination of liver sections, the livers were recovered from C57BL/6 mice 10 days following Ad vector administration. The livers were washed, fixed in 10% buffered formalin (Wako Pure Chemical Industries), embedded in paraffin, and processed for histology.
Albumin mRNA levels in the liver following Ad vector administration were determined by real-time RT-PCR using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan). The protocol for thermal cycling consisted of 60 seconds at 95 °C, followed by 40 cycles of 15 seconds at 95 °C and 60 seconds at 60 °C. The sequences of the primers were as follows: forward, 5′-TCC AAA CCT CCG TGA AAA CTA TG-3′; reverse, 5′-TGT GTT GCA GGA AAC ATT CGT-3′.
Albumin mRNA levels in the liver following Ad vector administration were determined by real-time RT-PCR using THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka, Japan). The protocol for thermal cycling consisted of 60 seconds at 95 °C, followed by 40 cycles of 15 seconds at 95 °C and 60 seconds at 60 °C. The sequences of the primers were as follows: forward, 5′-TCC AAA CCT CCG TGA AAA CTA TG-3′; reverse, 5′-TGT GTT GCA GGA AAC ATT CGT-3′.
4
Hepatic Lipid Quantification and Metabolic Markers
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Hepatic lipids were isolated as described previously.22 Total cholesterol, free cholesterol, triglycerides (TG) and free fatty acids were measured using Cholesterol E (catalog number 439‐17501), Free Cholesterol E (435‐35801), Triglyceride E (432‐40201) and Non‐Esterified Fatty Acid C (279‐75401) kits, respectively, according to manufacturer (Wako Pure Chemical Industries, Osaka, Japan) instructions. Serum glutamate‐pyruvate transaminase (GPT) levels were measured using a Transaminase CII Kit (431‐30901) according to manufacturer (Wako Pure Chemical Industries) instructions. Blood glucose was measured using an ACCU‐CHEK glucometer (Roche Diagnostics). Serum levels of insulin and VEGF were measured using enzyme‐linked immunosorbent assay kits (AKRIN‐031 and BMS619/2, respectively) according to manufacturer (Shibayagi, Gunma, Japan; eBioscience, San Diego, CA, USA) instructions.
5
Quantification of Liver Enzymes and Cytokines
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Plasma activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using the Transaminase CII kit (Wako Pure Chemical, Osaka, Japan) according to the manufacturer’s instructions, as previously described [26 (link)]. Plasma levels of tumor necrosis factor (TNF)-α (eBioscience, San Diego, CA, USA) were determined using commercially available ELISA kits, according to the manufacturer’s instructions. For relative quantification, calibration curves were prepared using standard solutions.
6
Biodistribution and Organ Toxicity of FA-NPs
7
Quantification of Liver Enzymes and Cytokine
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Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using a Transaminase CII kit (Wako Pure Chemical Industries, Osaka, Japan), as previously described [24 (link)–26 (link)]. Plasma levels of tumor necrosis factor alpha (TNFα) were measured using a commercially available ELISA kit (eBioscience, San Diego, CA, USA), as previously described [22 (link)]. For relative quantification, calibration curves were prepared using standard solutions.
8
Comprehensive Liver Function Evaluation
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Culture solution samples were collected in the culture circuit. Alanine aminotransferase (ALT) was analysed as an indicator of liver damage, and the bile secretion, albumin synthesis and urea production were analysed as indicators of liver physiological functions. The ALT concentration was measured by using the transaminase CII Kit (Wako). The albumin concentration was determined by the enzyme-linked immunosorbent assay (Rat Albumin ELISA Quantitation Set; Bethyl Laboratories, Montgomery, USA). Bile secretion was also measured by automatic collection through the catheter connected to the bile duct every 4 hrs. To analyse urea production, cleansing perfusion of the cultured liver was performed for 2 hrs using a Krebs-Henseleit buffer, and the liver was loaded with 1 mM ammonia for 30 min. Effluent buffer samples were collected every 3 min, and the urea concentrations in the supernatants were measured using the ammonia urea F-kit (JK International, Tokyo, Japan).
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