Beckman cytoflex flow cytometry

After treatment, THP-1 cells were washed and detached using Trypsin (Thermo Fisher Scientific). Subsequently, 1 × 10⁶ cells were resuspended in 100 µL of phosphate-buffered saline (PBS)/2% bovine serum albumin (BSA) solution and incubated with a CD86-PE (5 µL, 12-0869-42, clone: IT2.2, Thermo Fisher Scientific) or CD163-PE (5 µL, 333605, clone: GHI/61, BioLegend, San Diego, CA, USA) antibody for 40 min. After rinse twice, cells were resuspended in 500 µL of PBS/2% BSA solution. The cells were detected Beckman cytoflex flow cytometry (Beckman, USA). The Cytexpert Software was applied for the flow cytometry data analysis. The gating strategy for flow cytometry analysis in Figs. 3 and 6 was provided in Supplementary Figure no. 2.

Small interfering RNAs (siRNAs) (Table 1) were synthesized by GenePharma according to the sequence of erβ2 (GenBank Accession No GU721078.1). Transfection experiment was performed on an electroporation instrument NEPA21 (NEPAGENE, Chiba, Japan). Primary hepatocytes (1.5 × 10⁷ cells per mL) were suspended in the opti-MEM and mixed with siRNA at room temperature for 10 min. The mixture was transferred into electrode chamber and subjected to electroporation. The program was 195 V, 2.5 ms, 2 times, and 50 ms interval for poring pulse, and 20 V, 50 ms, 5 times, and a 50 ms interval for transfer pulse. After electroporation, the mixture was immediately transferred to L-15 medium containing 10% FBS in a 24-well plate. After preincubation at 28 °C for 24 h, the medium was removed and replaced with a fresh medium containing E₂. After 24 h incubation, cells were harvested for gene expression analysis. The electroporation experiment was performed in triplicate.
To analyze the transfection efficiency, siRNA-NC-Cy₃ (Cy₃-labeled negative control siRNA) was transfected into primary hepatocytes by electroporation according to the protocol above. After 24 h incubation, the fluorescence value was detected by Beckman Cytoflex flow cytometry (Beckman Coulter Inc., Brea, CA, USA), and analyzed by CytExpert software (Beckman Coulter Inc., Brea, CA, USA).