96 well transwell migration assay

CPCs were trypsinized, counted, and resuspended in starving medium that contained IMDM with GlutaMAX (ThermoFisher Scientific, Waltham, MA), 1% Insulin–Transferrin–Selenium (Life Technologies, Carlsbad, CA), and 0.5% FBS (Thermo Scientific, Waltham, MA). 50,000 CPCs were plated in the top chamber of a 96-well transwell migration assay (Corning, Union City, CA) with 8 μm pores. Standard CPC growth medium supplemented with 100 ng/mL of SDF-1α (Life Technologies, Grand Island, NY) was used in the bottom chamber as a chemoattractant. After 6 h, migrated cells in the bottom chamber were stained using Calcein AM (Fisher Scientific Pittsburg, PA) and quantified using a FLX800 fluorescent plate reader (Bio-Tek, Winooski, VT).

After exposure to 6–7 days of clinorotation, cells were trypsinized counted, resuspended in starving medium, and plated in the top chamber of a 96-well transwell migration assay (Corning, Union City, CA) with 8 micron pores. The transwell migration assay was performed according to manufacturer’s instructions (Corning, Union City, CA). In brief, cells were plated at a density of 50,000 cells per well in the top transwell chamber and growth medium supplemented with 100ng/mL of SDF-1α was used in the bottom chamber as a chemoattractant (Life Technologies, Grand Island, NY). After 6 hours, migrated cells in the bottom chamber were stained using Calcein AM (Fisher Scientific Pittsburg, PA) and quantified using a FLX800 fluorescent plate reader (Bio-Tek, Winooski, VT).