2030 arvotmx

Hepatocytes were incubated with 1-μg/mL PI (Sigma P4170) for about 30 min and observed under a DMi8 microscope (Leica Microsystems). The amount of LDH in the supernatant was measured using an LDH cytotoxicity detection kit (Takara MK401) following the manufacturer’s instructions. The amount of LDH in each sample was normalized against that of hepatocytes fully lysed with Triton X-100 (final 1% in PBS). The samples were individually mixed with reagents on microplates, and the absorbance was measured at 490 and 630 nm (as a control) using 2030 ARVO^TMX (Perkin Elmer) or Multiskan GO (Thermo Fisher Scientific) after a 30-min incubation at room temperature.

Macrophage-mesangial cell binding assay was performed using the Vybrant cell adhesion assay Kit (Life technologies, Carlsbad, CA) according to the manufacturer’s instruction. Briefly, Mes13 cells or HUVEC transfected with siRNA against CTGF or control were cultured for 24 h in 96-well plates before pretreatment with 10 ng/ml TNF-α. Twenty-four hours after TNF-α stimulation, Mes 13 cells or HUVEC were treated with 100 ng/ml human recombinant CTGF for 3 hours, and then were co-cultured with RAW264.7 cells (5 × 10⁶ cells/ml) which were pretreated with 5 μM calcein-bis (carboxymethyl) aminomethyl (AM) solution in DMEM for 30 min. Two hours after co-culture, cells were washed four times with phosphate-buffered saline to remove non-adherent calcein-AM labeled Raw264.7 cells. Fluorescence intensity was measured using a 2030 ARVO^TM X (PerkinElmer, Waltham, MA) at excitation and emission wavelengths of 485 nm and 535 nm, respectively.