Premix wst 1 cell proliferation assay

Cells (3.0×10⁴ cells per well) were placed into 500 μl of medium in 24-well plates (Corning Inc., New York, NY, USA). After culturing for 24, 48, 72, 96 or 120 h, the supernatant was removed, and cell-growth inhibition was analyzed using the Premix WST-1 cell proliferation assay (TAKARA Bio, Otsu, Japan) according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader (Perkin Elmer Inc., Waltam, MA, USA). All assays were carried out in triplicate.

To evaluate cell morphology, GO-treated THP-1 cells were gently washed and then images captured by light optical microscopy (CK70, Olympus, Tokyo, Japan). Cell viability was evaluated using a PreMix WST-1 cell proliferation assay (TaKaRa Bio, Shiga, Japan) according to the manufacturer’s protocol26 27 (link). Membrane integrity was assessed using a LDH Cytotoxicity Detection kit (TaKaRa Bio) according to the manufacturer’s protocol26 27 (link). We additionally confirmed membrane integrity by acridine orange (Sigma-Aldrich) staining. Briefly, cells were washed three times with pre-warmed DPBS and then stained with 20 μg/mL acridine orange in pre-warmed DPBS for 15 min. Thereafter, the cells were washed and filled with DPBS prior to visualization using a fluorescence microscope (Axio Observer D1, Carl-Zeiss, Oberkochen, Germany). Apoptotic cell death, and ROS and IL-1β production were measured as described previously26 .

MM cells (5 x10³) were treated for 48 h with CP at the indicated concentrations in 96-well plates. Subsequently, the inhibitory effect of CP on MM cell line growth was assessed using a WST-1 assay (Premix WST-1 Cell Proliferation Assay, Takara Bio, Otsu, Japan) and an Infinite M1000 PRO microplate reader (Tecan Japan, Kawasaki, Japan) as described previously [34 (link)]. The half-maximal inhibitory concentration (IC₅₀) was defined as the drug concentration resulting in 50% cell survival relative to that of DMSO-treated cells. To analyze the proliferation of MM cells with or without BMSCs, we used a BrdU assay (BrdU cell proliferation assay reagent, Cell Signaling Technology, Danvers, MA) [5 (link)]. Initially, this assay was established using BrdU incorporated into cellular DNA during cell proliferation, and it is similar to the ³H-incorporation assay but does not use radioactive reagents. Therefore, the BrdU assay is suitable for this proliferation assay.
Apoptosis was evaluated by PARP, caspase-3, caspase-8, and caspase-9 western blotting and quantified using an Annexin V/7-AAD staining kit (BD Biosciences, San Jose, CA), as per the manufacturer’s instructions, followed by analysis on a BD FACS Canto II using FACSDiva (BD Biosciences, Tokyo, Japan) [35 ].