How to prepare vitronectin substrate plates for pluripotent stem cell culture

This video will show you how to coat culture plates with vitronectin, so you will be ready to transfer newly forming iPSC colonies or frozen cultures to Essential 8 Medium. [AUDIO TRANSCRIPT ] This video will show you how to coat culture plates with vitronectin, so you will 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 vitronectin-coated plates. The optimal working concentration of vitronectin is cell-line dependent and must be determined empirically. We recommended using a final coating concentration of 0.1--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. Prior to coating culture vessels, calculate the working concentration of vitronectin using the following formula and dilute the stock appropriately. The product manual for vitronectin contains a list of culture dish surface areas and volume of vitronectin required. As an example,, to coat a 6-well plate at a coating concentration of 0.5 μg/cm2, you'll need to prepare 6 mL of diluted vitronectin solution (10 cm2/well surface area and 1 mL of diluted vitronectin/well). Upon receipt, thaw the vial of vitronectin at room temperature and prepare 60μL 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 60μL aliquot of vitronectin from --80°C storage and thaw at room temperature. You'll need one 60μL aliquot per 6-well plate. Add 60 μL of thawed vitronectin into a 50-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, a 1:100 dilution Add 1 mL of the diluted vitronectin solution to each well of a 6-well plate. When used to coat a 6-well plate (10 cm2/well) at 1 mL/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 one week. Do not allow the vessel to dry. Prior to use, pre-warm the culture vessel to room temperature for at least 1 hour.