Infinite 4300

4T1 murine breast carcinoma cells were seeded in 96 well cell culture plates (Corning Costar) at 5,000 cells per well. Cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum. MU was solubilized in dimethyl sulfoxide (DMSO), diluted into RPMI 1640 media supplemented with insulin, transferrin, and sodium selenite (ITS, Sigma)), and added to cells. MU-P was solubilized directly into RPMI-ITS and added to cells. Twenty-four hours after plating, cells were washed 3x with phosphate buffered saline (2.7mM KCl, 1.5mM KH₂PO₄, 136.9mM NaCl, 8.9mM Na₂HPO₄, pH 7.4; PBS). MU and MU-P were added to cells after washing with or without Phosphatase Inhibitor Cocktail 2 (Sigma). Forty-eight hours later, 50 μL of media was removed and frozen. Fifteen μL of alamarBlue (Life Technologies) was added to cell media and incubated for 3 hours. Cell number was quantified by measuring alamarBlue fluorescence at 585 nm after excitation at 570 nm using a Tecan Infinite 4300 (Tecan Group Ltd).
HA levels in culture media were quantified by using the HA DuoSet ELISA assay (R&D Systems). Frozen media samples were thawed and diluted 1:100 in Reagent Diluent (R&D Systems). The ELISA was run according to the manufacturer’s specifications. Total HA in culture media was normalized to cell number using the alamarBlue assay and to DMSO control wells.

Solvents were removed under reduced pressure using a rotary evaporator. Fluorescence spectroscopy was measured on a FluoroLog-3 spectrofluorimeter (Horiba Jobin Yvon) equipped with a temperature-controlled stage (LFI-3751) or using a Tecan Infinite 4300 (Tecan Group Ltd). Data acquisition was done through FluorEssence software (Horiba Scientific). High-pressure liquid chromatography (HPLC) was performed on an Agilent 1100 HPLC (Agilent).