Agilent wave software

The Seahorse XF Mito Stress Test (Agilent, 103015) was performed using the Seahorse XFe96 Analyzer. For melanoblasts and melanocytes, cells 30,000 melanoblasts and 10,000 melanocytes were seeded per well in an XF cell plate as previously described^{13 (link)}. For human A375 cells, cells were treated with DMSO, IBMX, or Forskolin as described above then cells were trypsinized and resuspended in drug-containing media at 30,000 cells per well in an XF cell plate coated with 0.05% poly-L-lysine then incubated overnight (Sigma-Aldrich, 4707).
Cells were incubated in XF Mito Stress Test assay medium (Seahorse XF DMEM medium, pH 7.4, 10 mM glucose, 2 mM glutamine, 1 mM sodium pyruvate) for 1 h prior to measurement in a CO₂ free incubator at 37 °C. During assay run, cells were exposed to 2.0 µM oligomycin, 2.0 µM FCCP, and 0.5 µM rotenone/antimycin A. OCR and ECAR were normalized to nuclei fluorescence unit (FU) via SYTO 24 (Thermo Fisher, S7559) or protein via Pierce BCA Protein Assay Kit (Thermo Fisher, 23227) as indicated. Experimental measurements were analyzed using the Agilent Wave software.

Flux analysis data (technical replicates) of each measurement were normalized in the Agilent Wave Software (Agilent Seahorse Technologies) and exported as mean value to the Multi-File Seahorse XF Cell Energy Phenotype Test Report Generator. Standard deviation of a threefold approach (biological replicates) was then calculated with OriginPro 2017G. Data are shown as mean value with SD and were analysed by GraphPad software using repeated measures one-way ANOVA with Tukey’s post-hoc for flux analysis and t-test for quantification of cell and microcarrier parameters via immunofluorescence analysis. In experiments without drug application, control is defined as cells without application of microcarriers (no-carrier control). For the application of nocodazole, cell samples with microcarriers incubated in maintenance medium (maintenance control) or the solvent DMSO (solvent control) were employed as controls. * p < 0.05, ** p < 0.01, *** p < 0.001.

Mitochondrial respiration, as a major energy-producing pathway for cells, was measured by an Agilent Seahorse XFp extracellular flux analyzer, as described previously in [24 (link)]. The Seahorse XF Cell Mito Stress Test Kit (Agilent, Santa Clara, CA, USA) reports multiple key parameters, including basal respiration, ATP-linked respiration, and spare respiratory capacity. To analyze metabolic activity by means of the Seahorse platform, cells were incubated in a 24-well plate at a concentration of 1.5 × 10⁵ cells per well; pseurotin D (1–25 µM) was added and incubated for 24 h at 37 °C under a 5% CO₂ atmosphere. After the incubation period, cells were transferred into a poly-l-lysine coated 8-well plate (Agilent Seahorse XFp miniplate). Attached cells were washed with the assay medium, and a final volume of the assay medium was added and measured. The total amount of cells was used for data normalization. The Agilent Wave software was used for data evaluation. From these characteristic kinetic curves, basal respiration, proton leak, maximal respiration, and non-mitochondrial respiration were determined.