Xtt assay kit

Cytotoxicity of biosynthesized CuO-NPs was assessed by XTT assay (2,3-bis[2-methoxy-4-nitro-5-sulfoxyphenyl]-2H-tetrazolium 5-carboxyanilide inner salt), against NIH 3T3 fibroblast cells using XTT assay kit (Roche, Switzerland) as described by ref. 47 (link). Cultured fibroblast cells when reached at 85% confluency were trypsinized (using 1X trypsin-EDTA solution) and were seeded in 96 well plate (3000 cells per well) providing the complete growth medium, Dulbecco's Modified Eagle Medium Low Glucose (DMEM-LG) supplemented with 20% fetal bovine serum (FBS). The plate was incubated at 37 °C in the 5% CO₂ incubator for 24 h. After overnight incubation the cells were treated with 20 μg mL⁻¹, 50 μg mL⁻¹, 75 μg mL⁻¹ and 100 μg mL⁻¹ concentration of CuO-NPs suspended in serum free low LG medium for 24 h. The group including cells seeded on simple tissue culture plate without any treatment of CuO-NPs is considered as control. After 24 h treatment, the wells media was aspirated and washed twice with 1XPBS. 50 μL of fresh prepared mixture of XTT (with electron coupling reagents prepared in ratio of 50 : 1) was added to corresponding wells of plate. The plate was then wrapped completely in aluminum foil and kept in 5% CO₂ incubator at 37 °C. The absorbance was recorded at wavelength of 450 nm with 630 nm as a reference wavelength. The experiment was repeated twice in a triplicate manner.

The distilled water extract of DM was purchased from the Korean Plant Extract Bank (Daejeon, Korea). The TRAP staining solution was purchased from Sigma Aldrich (St. Louis, MO, USA), and the XTT assay kit was obtained from Roche (Indianapolis, IN, USA). The α-minimum essential medium (α-MEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Gibco-BRL (Grand Island, NY, USA); soluble human recombinant M-CSF and RANKL were purchased from Peprotech (London, UK). Specific primary antibodies against phospho-p38 (#9211), p38 (#9212), phospho-JNK (#9251), JNK (#9252), phospho-Akt (#9271), Akt (#9272), and phospho-IκB (#2859) were purchased from Cell Signaling Technology (Beverly, MA, USA), while phosphor-Btk (GTX61791) was purchased from GeneTex (Irvine, CA, USA) and β-actin (A5441; housekeeping gene) was obtained from Sigma Aldrich. Specific antibodies against phospho-PLCγ2 (sc-101785), PLCγ2 (sc-5283), IκB (sc-371), and c-Fos (sc-7202), and NFATc1 (sc-7294) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The flushing was continued for 3 days and on the next day of the last flushing, viabilities of the cells were measured using the XTT assay kit (Roche Diagnostics GmbH, Mannheim, Germany). From the 4 replicates flushed with medium without any drug, the mean extinct at 450 which reflects the viability of the cells was calculated and used to normalize all other extinct values. Using the normalized values, the mean and standard deviation of the viability of the cells flushed with each drug at each concentration were calculated.
For the cells of the 4th donor, we also included a treatment with B-OT permanently in the medium parallel to the wash treatment, both for 1, 2 and 3 days.

To perform clonogenicity assay [13 (link)], breast cancer cells were treated with 100 ng/ml leptin and 5μM HNK alone or in combination as indicated for 10-days; colonies were counted. Anchorage-independent growth of breast cancer cells in the presence of leptin and/or HNK was assayed by colony formation in soft agar [27 (link)]. Cell viability assay was performed using a commercially available XTT assay kit (Roche Applied Science, Indianapolis, IN).