Cryopreservation I agree totally with Haywood Pyle. Firstly, cleaning up the cell preparation, for example on Ficoll-Paque, is advantageous, particularly when dealing with leukocytes. Adding the DMSO dropwise with GENTLE agitation in an ice bath is essential for subsequent efficient recovery of the cells. Rapid thawing is also essential, with holding the cells at <6oC until the DMSO has been removed by washing the cells. We have found a few extra tricks which help, in particular with leukocyte preparations:- - the serum component of your freezing medium should also be added dropwise with gentle shaking in an ice bath, before adding the DMSO (also dropwise); - the cells should be stored at 1-7 X 10(7)/ml in 1ml aliquots; - when thawing (we do this with shaking in a 37oC water bath for efficiency), watch for the last piece of ice disappearing, at which point the temperature within the amp will be 0-4oC; - when diluting the cells with ice-cold medium or buffer (we actually use Ca2+Mg2+ free PBS containing 0.03mM EDTA to prevent clumping), add this slowly with constant GENTLE shaking to the cells. The latter step of slowly adding the diluting buffer to the thawed cells seems to be at least equally important to the slow addition of the DMSO before freezing, particularly when dealing with leukocytes. One word of negativity - freezing and subsequent successful recovery of mature granulocytes is a nightmare, even with this method. Have fun! Ken McCullough IVI, CH-3147 Mittelhausern, Switzerland Tel. (+41) 31 848 9361 Fax (+41) 31 848 9222 email: kenneth.mccullough@ivi.admin.ch
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