How to prepare vitronectin substrate plates for pluripotent stem cell culture

This video will show you how to coat culture plates with matrices to support cell attachment and growth, so you'll be ready to transfer newly forming IPSC colonies or frozen cultures to Essential 8 Medium. Before using the Essential 8 complete medium, you'll need to prepare matrix-coated plates. For this video we are using vitronectin, but other matrices can be used with a similar procedure. The optimal working concentration of vitronectin is cell line dependent and must be determined empirically. We recommend using a final coating concentration of 0.1 to 1.0 µg/cm2 on the culture surface depending on your cell line. We routinely use vitronectin at 0.5 µg/cm2 for human PSC culture. Upon thawing the vitronectin vial, it should be mixed by gentle inversion to ensure homogeneity ahead of aliquoting. This is important to make sure each of the aliquots has the same protein concentration. Prior to coating culture vessels, calculate the working concentration of vitronectin. We recommend 0.5 µg/cm2 with 1 mL being added per well of 1:100 dilution in DPBS (-/-). Upon receipt, thaw the vial of vitronectin at room temperature and prepare 60 µL aliquots of vitronectin in polypropylene tubes. Freeze the aliquots at -80˚C or use immediately. To coat the wells of a 6-well plate, remove a 60 µL aliquot of vitronectin from -80˚C storage and thaw at room temperature. You'll need one 60 µL aliquot per 6-well plate. Add 60 µL of thawed vitronectin into a 15 mL conical tube containing 6 mL of sterile DPBS without calcium and magnesium at room temperature. Gently re-suspend by pipetting the vitronectin dilution up and down. This results in a working concentration of 5 µg/mL at 1:100 dilution. Add 1 mL of the diluted vitronectin solution to each well of the 6-well plate. When used to coat a 6-well plate at 1 mL per well, the final concentration will be 0.5 µg/cm2 If you're using a different type of culture vessel refer to the vitronectin product insert for detailed information on volumes. Incubate the coated plates at room temperature for 1 hour. Note, the culture vessel can now be used or stored at 2-8˚C wrapped in laboratory film for up to 1 week. Do not allow the vessel to dry. One method to prevent drying is to make a 1:200 dilution, and then adding 2 mL for coating. Prior to use, pre-warm to culture vessel to room temperature for at least 1 hour.