Trypanosoma cruzi Chagas
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ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
Additional information on this culture is available on the ATCC web site at www.atcc.org.
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1. Agitate a culture at or near peak density by inverting several times.
2. Aseptically transfer a 0.1 ml aliquot to a T-25 tissue culture flask containing 10 ml of fresh complete medium.
3. Screw cap on tightly and incubate at 20-25°C.
4. Subculture every 14-21d.
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.
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Dvorak JA, et al. Trypanosoma cruzi: flow cytometric analysis. I. Analysis of total DNA/organism by means of mithramycin-induced fluorescence. J. Protozool. 29: 430-437, 1982. PubMed: 6182288
Bongertz V, Dvorak JA. Trypanosoma cruzi: antigenic analysis of cloned stocks. Am. J. Trop. Med. Hyg. 32: 716-722, 1983. PubMed: 6349399