Seakem le agarose 50004

To establish spheroids that finally consist of 5000 cells/spheroid, cells were suspended in DMEM at 50,000 cells/mL and 100 μL of this suspension was plated per well in a 96-well plate coated with 1.2% agarose (SeaKem LE Agarose 50004, Lonza, USA) to prevent cell adhesion. Cells were incubated for 4 days to allow for compact spheroid formation before proceeding with the experiments described below.

The rhabdomyosarcoma (RD and RH-30) cells were plated in a 96-well
plate that was coated with 1.2% agarose (SeaKem LE Agarose 50004,
Lonza, USA) to prevent cell adhesion. The cells were subsequently
incubated under physiological conditions for 2 days to enable compact
spheroid formation. Thereafter, the spheroids were treated with the
ruthenium(II) complex of interest at 2× IC₅₀, IC₅₀, and ¹/₂IC₅₀ concentrations,
and the vehicle (0.1% DMSO) and cisplatin (at IC₅₀) were
included as controls. Images of the RMS spheroids were taken, using
an EVOS M5000 Imaging System microscope, to monitor spheroid growth
over 6 days. Three independent experiments with at least four replicates
for each condition were performed. The area of the spheroids was measured
using ImageJ v1.50i software.^{105 (link)}