induced pluripotent stem cells

this video will show you how to coat cultureplates with vitronectin, so you will be ready to transfer newly forming ipsc colonies orfrozen cultures to essential 8â„¢ medium. before using the essential 8â„¢ complete medium,you'll need to prepare vitronectin-coated plates. the optimal working concentrationof vitronectin is cell-line dependent and must be determined empirically. we recommendedusing a final coating concentration of 0.1–1.0 âµg/cm2 on the culture surface, dependingon your cell line. we routinely use vitronectin at 0.5 âµg/cm2 for human psc culture. prior to coating culture vessels, calculatethe working concentration of vitronectin using the following formula and dilute the stockappropriately. the product manual for vitronectin

contains a list of culture dish surface areasand volume of vitronectin required. as an example,, to coat a 6-well plate at a coatingconcentration of 0.5 âµg/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 vitronectinat room temperature and prepare 60âµl aliquots of vitronectin in polypropylene tubes. freezethe aliquots at –80â°c or use immediately. to coat the wells of a 6-well plate, removea 60âµl aliquot of vitronectin from –80â°c storage and thaw at room temperature. you’llneed one 60âµl aliquot per 6-well plate. add 60 âµl of thawed vitronectin into a 50-mlconical 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 of5 âµg/ml, a 1:100 dilution add 1 ml of the diluted vitronectin solutionto each well of a 6-well plate. when used to coat a 6-well plate (10 cm2/well) at 1ml/well, the final concentration will be 0.5 âµg/cm2. if you’re using a different type of culturevessel, refer to the vitronectin product insert for detailed information on volumes. incubate the coated plates at room temperaturefor 1 hour.

note: the culture vessel can now be used orstored at 2–8â°c wrapped in laboratory film for up to one week. do not allow the vesselto dry. prior to use, pre-warm the culture vessel to room temperature for at least 1hour.