Qubit 3.0 fluorometer

To further investigate the role of HA35 in regulating gene expression levels in immune cells, RNA sequencing was conducted on RAW264.7 cells and BV2 cells to identify transcriptional changes associated with HA production regulation. RAW264.7 cells and BV2 cells were inoculated in 6-well plates at a density of 1 × 10⁶ cells/mL and cultured overnight. After adhesion, the cells were replaced with serum-free DMEM and MEM, and then resuspended in serum-free RPMI 1640. One milliliter of 300 µg/mL HA35 or HA1600 was added to the respective culture systems and incubated at 37 °C for 6 h. Post-incubation, the cells were detached using a cell scraper and transferred to RNase-free EP tubes. RNA was extracted using 500 µL of TRIzol solution per sample to prepare for transcriptome sequencing. The extracted total RNA samples were analyzed using 1% agarose gel electrophoresis, a Nanodrop 2000 spectrophotometer, a Qubit^® 3.0 Fluorometer, and an Agilent 2100 Bioanalyzer (all from Thermo Fisher Scientific, Waltham, MA, USA). Following transcriptome sequencing, library preparation, and quality verification, sequencing was performed primarily using the Illumina HiSeqTM X TEN system [32 (link),33 (link),34 (link)].

Huh7, MHCC-97H, and SNU449 cells treated with 0.5 μM flubendazole or DMSO for 72 h were harvested using TRIzol and subjected to mRNA Sequence Analysis (Sinotech Genomics, Shanghai). Total RNA was purified using the Tianmo#TR205-200 kit. The quality of total RNA was determined by Agilent Bioanalyzer 2100 (Agilent technologies, California, USA) and quantified by Qubit®3.0 Fluorometer and NanoDrop One Spectrophotometer. Enrichment of differential gene analysis for KEGG and gene ontology (GO) pathways was performed using GSEA software.