Crystal violet assay kit

The Crystal Violet Assay Kit (#ab232855; Abcam, Cambridge, UK) was used for cell cytotoxicity and cell viability studies, as previously described [22 (link)]. Briefly, the HNSCC cells were plated 3000–4000/well in 96-well plates and treated with hDT806, cetuximab (#A2000; Selleckchem, Houston, TX, USA), or erlotinib (#S1023; Selleckchem) following a 2-fold serial dilution. Five to seven days later, a crystal violet staining assay was performed according to the manufacturer’s instruction to determine cytotoxicity and cell viability. Optical density (O.D.) of each well was measured at 595 nm on a microplate reader. The percentage of viable (attached) cells against the values of untreated control samples were calculated to represent cell viability [23 (link)].

Metabolic analysis was measured with Seahorse XFe24 analyzer using Seahorse XF substrate oxidation stress kits for glucose/pyruvate (Agilent, 103,673-100). To understand the changes in cardiomyocyte metabolism, adult WT and CTRP9-KO cardiomyocytes were isolated with Langendorff perfusion; 12,000 cardiomyocytes were then plated on Seahorse 24-well FluxPak plates and cultured for 24 h. On the next day, culture medium of the cells was exchanged with Seahorse XF DMEM (Agilent, 103,575-100), including 1 M glucose (Agilent, 103,577-100), 100 mM pyruvate (Agilent, 103,578-100) and 200 mM glutamine (Agilent, 103,579-100) and incubated in a non-CO₂ incubator at 37 °C for 1 h. For experimental groups UK5099 (2 μM) inhibitor was loaded into the cartilage, while the control groups were treated with assay medium alone. Previously optimized amounts of oligomycin, FCCP and rotenone/antimycin A were also loaded into the cartilage. Substrate stress-test assays were run according to the manufacturer’s protocol and analyzed using Seahorse Analytics web-based software. Data were normalized to the number of cells, which was assessed with colorimetric measurement after staining with crystal violet assay kit (Abcam, Cambridge, UK, ab232855).

Abcam’s Crystal Violet Assay Kit (cell viability) (ab232855) and MTT reagents were used for cell viability assay. RAW 264.7 cells (5 × 10⁴ cells/well) were seeded into 96-well plates and left to stabilize in a 37 °C incubator for 24 h, followed by treatment with the EUFOC (0.05, 0.1, 0.3, 0.5, 1.0, or 2.0%).
The MTT solution was added after 24 h to confirm cytotoxicity. The sample was removed after 2 h and 150 μL of DMSO was added to each well to dissolve the formed formazan, followed by the measurement of absorbance at 540 nm.
MTT was used to confirm the cytotoxicity of the EUFOC in keratinocytes. HaCaT cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (PS, Gibco, Grand Island, NY, USA) under 5% CO₂ at 37 °C. The cells were seeded in 96-well plates at a density of 2 × 10⁴/well and then stabilized for 24 h. The EUFOC, diluted in serum-free medium, was further diluted with ethanol (70%) to prepare different concentrations of the sample. After 24 h, the medium was removed from the cells and 20 µL of MTT (5 mg/mL) was added, followed by incubation in a cell incubator (37 °C, 5% CO₂) for 2 h. Next, MTT was removed, and 100 μL of DMSO was added. After ensuring that the crystals were dissolved in the stirrer, the absorbance was measured at 540 nm.