Since the bone marrow express proteins help in the adhesion of HSCs and leukemic stem cells (LSCs), it was important to coat the 3-D scaffolds with proteins. Protein adsorption was performed by incubating different blends of scaffolds overnight with 20% FBS or with 20 μg/mL FN in PBS. The scaffolds were wetted before protein coating by incubating the scaffolds in 70% ethanol for 1 minute, followed by washing with PBS three times. Subsequently, the scaffolds were incubated in a protein solution for 12 hours and washed twice with PBS by gentle agitation to remove non-adsorbed or weakly adsorbed proteins. The protein-coated scaffolds were then incubated in elution buffer (0.025% SDS and 0.0025% CHAPS) to extract the proteins adsorbed on these scaffolds. To estimate the concentration of total protein adsorbed on the scaffolds, one volume of eluted solution was incubated with eight volumes of BCA reagent for 30 minutes at 37°C. In order to estimate the FN concentration in the extract, the scaffolds were incubated with an equal volume of micro BCA reagent for 2 hours at 37°C. Optical density was recorded at 562 nm using a BioTek PowerWave™ XS plate reader (BioTek Instruments Inc, Winooski, VT, USA).28 (link)