T. gondii - The RH strain used was obtained from animal facilities of our laboratory. The tachyzoites were cultured by intraperitoneal inoculation of female Balb/C mice. The parasites were harvested from mice by rinsing of the peritoneal cavity with 5 ml of Eagle modified minimum essential medium (MEM).
<br>
<br>
TG180 murine sarcoma cell cultures - The TG180 cell line was derived from the ATCC (CCRFS-180 II) sarcoma 180 in 1958 (Sartorelli & Booth 1961). A sub-population of this cell line adapted to in vitro culture (Couatarmanach et al. 1991) was used in our experiments. For the in vitro experiments the cells were grown in MEM supplemented with 10% fetal calf serum, 40 킽/ml gentamycin and 25 킽/ml amphotericin B. The cultures were incubated at 37oC in an atmosphere of 95% air and 5% CO2 in 5 ml-tissue culture flasks. The cells were observed daily by inverted light microscopy, growing in suspension as non-adherent polymorphic cells.
<br>
<br>
Then, 3.0x106 TG180 cells were inoculated into flasks containing 5 ml of MEM culture medium and used after 8-14 passages. Cell countings were made with the use of "Trypan blue".
<br>
<br>
For in vivo experiments, TG180 sarcoma murine cells were grown as ascitic tumors in adult female Balb/C mice in our animal facilities. For the experiments, mice were intraperitoneally inoculated with 1.0x106 TG180 cells.
<br>
<br>
In vitro infection - Six culture flasks were infected with 1.0x106 tachyzoites each. This 3:1 TG180:T. gondii ratio was used as it has been already shown to yield large numbers of intracellular parasites (Couatarmanach et al. 1991). Cell suspensions were collected from the flasks daily from the first to the sixth day post-infection by centrifugation at 1,000g for 10 min. To avoid cell damage due to medium acidification, MEM culture medium was added on the second and third day post-infection. Cell suspensions were fixed overnight with 2.5% glutaraldehyde/2% paraformaldehyde (Karnovsky 1965) diluted in 0.1M phosphate buffer, pH 7.2, washed three times in 5 ml of phosphate buffered saline (PBS) pH 7.4 and processed for transmission electron microscopy as described below. On day 6, culture flasks of uninfected cells were also fixed and used as controls.
<br>
<br>
In vivo infection - Female Balb/C mice were intraperitoneally inoculated with a suspension of 1.0 x 106 TG180 cells. Subsequently, these animals were also inoculated with another cell suspension containing 1.0 x 106 T. gondii by the same route. Mice were sacrificed after 10, 20, 30 min and 24 and 48 h post-infection and their ascitic fluids were collected. This material was fixed with 2.5% glutaraldehyde/2% paraformaldehyde, washed three times in PBS and processed for transmission electron microscopy as described below.
<br>
<br>
Transmission electron microscopy - Briefly, the material was post-fixed for 1 h in 1% OsO4, dehydrated in increasing concentrations of ethanol and embedded in Epon. Ultrathin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate and examined in a Zeiss EM 109 transmission electron microscope.
<br>
<br>
<!-- -->
