2.6 Total protein versus cell mass as a normalization strategy
In this study, metabolites were quantified based on the weight of total protein. Single cell suspensions from both 2D monolayer culture and 3D MTS were centrifuged for 10 min at 12,000×g at 4℃. For measurement of total protein amount, the pellets were resuspended in Radio Immunoprecipitation Assay (RIPA) Lysis Buffer (Thermo Scientific™, 89901, USA). The protein concentration was determined using a bicinchoninic acid (BCA) assay (Thermo Fischer Scientific, Waltham, MA, USA) following the instructions of manufacturer. 5 μL each protein lysate was added to 2 μL reagent A, followed by the addition of 100 μL reagent B. After 30 min at 37℃, absorbance was read at 562 nm by fluorescence microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentrations in the sample were determined using standard curves generated by linear regression analysis.