Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.
2.?? Aseptically transfer the cell suspension to 15 ml plastic centrifuge tubes.
3.?? Centrifuge at ~800 x g for 5 min.
4.?? While cells are centrifuging, prepare a 10% solution of DMSO in complete ATCC Medium 1029.? Cool on ice.
5.?? Remove the supernatant and pool the cell pellets to the final volume desired with fresh growth medium.
6.?? Combine the cell suspension with an equal volume of 10% DMSO cryoprotectant solution (prepared in step 4) to yield a final concentration of 5% 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.? Alternatively, place the vials in a Nalgene 1°C freezing apparatus.? Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately -1°C/min.). ?
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 complete ATCC medium 1029.
12.Screw the cap on tightly and incubate at 20-25°C.??? Observe the culture daily and transfer when numerous trophozoites are observed.?????????
Walton BC, et al. The isolation and identification of Trypanosoma cruzi from raccoons in Maryland. Am. J. Trop. Med. Hyg. 7: 603-610, 1958. PubMed: 13595203
Diamond LS, Rubin R. Experimental infection of certain farm mammals with a North American strain of Trypanosoma cruzi from the raccoon. Exp. Parasitol. 7: 383-390, 1958. PubMed: 13562102
Clark CG, Pung OJ. Host specificity of ribosomal DNA variation in sylvatic Trypanosoma cruzi from North America. Mol. Biochem. Parasitol. 66: 175-179, 1994. PubMed: 7984184
