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here we will demonstrate how to expand human airway epithelial cells and initiate cultures at the air liquid interface or ali the ali workflow involves three phases expansion culture in a flask submerged expansion in inserts and ali culture in inserts in this video we will focus on phase one and two the two expansion steps of the workflow phase one expansion culture in a flask to begin thon yuma called ex 50x supplement at room temperature if a precipitate is observed after thawing mix the contents by inverting the vial a couple of times the precipitate will return to solution and will not affect the culture performance use the supplement immediately or aliquot and store at minus 20 degrees celsius prepare 500 milliliters of complete pneumococ ex medium mix the components thoroughly the complete medium can be stored at two to eight degrees celsius for up to four weeks to initiate culture obtain frozen primary airway cells such as bronchial tracheal nasal or small airway epithel
