Sulforhodamine-B (SRB) Assay

For these studies, we plated 2 × 10³ HNSCC cells per well (UM-SCC-11B or UM-SCC-22B, kindly provided by Dr. T. Carey) and exposed them to 0.01 to 100 μg/ml MEDI5117 or 100 μg/ml control IgG (R347). After 24 to 72 hours, cells were fixed with 50% trichloroacetic acid and stained with 0.4% sulforhodamine-B solution (SRB; Sigma Aldrich). Unbound SRB dye was removed by washing with 1% acetic acid. Plates were air-dried and bound SRB was resolubilized in 10 mM unbuffered Trizma base. Absorbance was analyzed on a microplate reader at 560 nm (Genios Tecan). Test results were normalized against initial plating density and IgG controls. Quadruplicate wells per condition were evaluated and are representative of at least two independent experiments.