1.?? Harvest cells from several cultures which are in logarithmic to late stationary phase of growth.? Vigorously agitate to suspend the cells.
2.?? Aseptically transfer the cell suspension to 15 ml plastic centrifuge tubes.
3.?? Centrifuge at 200 x g for 5 min.
4.?? While cells are centrifuging, prepare a 20% solution of DMSO in ATCC Medium 2375.
5.?? Remove the supernatant and pool the cell pellets into a final volume of 4.5 ml.
6.?? Combine the cell suspension with an equal volume of 20% DMSO cryoprotectant solution (prepared in step 4) to yield a final concentration of 10% DMSO.
7.?? Dispense in 0.5 ml aliquots to 1.0-2.0 ml Nunc vials (special plastic vials for cryopreservation).
8.?? Place the vials in a controlled rate freezing unit.? From room temperature cool at -1°C/min to -40°C.? At -40°C, plunge ampules into liquid nitrogen.
9.?? Store ampules in a liquid nitrogen refrigerator until needed.
10.????????? To establish a culture from the frozen state, place a frozen ampule in a 35°C water bath just enough to cover the frozen material.? Allow the ampule to thaw completely (2-3 min).
11.????????? Immediately after thawing, aseptically remove the contents and transfer to a T-25 tissue culture flask containing 10 ml of fresh ATCC medium 2375.
12.Screw the cap on tightly and incubate at 25°C.? Observe the culture daily.? Transfer the culture when many trophozoites are observed.?????????
