we are working with cultures of T. cruzi epimastigotes that overexpress a protein of interest. In several attempts to obtain cell-derived trypomastigotes, we infected VERO with metacyclics (differentiated in vitro using TAU or blood-agar medium), but the parasites that emerged after the cell lysis almost immediately acquiere an amastigote-like morphology, and only few remain as trypomastigotes (less than 1%). This prevents us from obtaining transgenic cell-derived trypomastigotes. Has anyone had this problem? Some advice to overcome this, which exclude infecting animals? Thanks a lot!
Dear Mariana
I think that you should check the pH of the medium. One of the most important factors for amastigote differentiation is an acidic medium. Some technical issues:
1) Reduce the rate of parasite/cell in the infection.
2) Change the harboring cell.
3) Add buffers to delay the reduction of pH for high metabolic rate.
I hope to help.
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