Inhibition of CTK11 growth using exogenous thymidine. Protocol: JRadke060601 Ó2001 Michael W. White-Department of Veterinary Molecular Biology || questions@montana.edu
WhiteProtocols2001
1.0 Overview. CTK11 growth can be arrested at the G1-S boundary using exogenous thymidine in the growth media. Tachyzoites that have completed S-phase under the effects of thymidine are able to transit mitosis, cytokinesis and most of G1 before reaching the G1-S block. Four-hour treatment (10 mM thymidine) appears optimal, but limits the effective synchrony to a 2 h window within the parasite cell cycle. Thymidine is a known mutagen and treatment beyond 6 h (10 mM) kills » 20% of the population while longer treatment or higher doses become increasingly toxic. The growth of parental RH tachyzoites is unaffected by these compounds at doses > 1 mM. Following release from the thymidine block, the arrested population will move into S phase (< 2 h), transit G2+M and reach G1 of the subsequent cell cycle beginning » 5 h post release. As observed in mammalian cell systems, synchrony is progressively lost as the population traverses the next cell cycle. 2.0 CTK11 Tachyzoite Growth and Synchronized Replication. 2.1 Inoculate a single HFF confluent T-75 cm^2 flask with 10x10^6 tachyzoites in standard growth media (1% DMEM, dialyzed* FBS, P/S, AmphB/*NOTE: CTK11 tachyzoites should also be cultured in media made from dialyzed FBS) and allow this population to invade cells for 1 h. Exchange the media 1X to remove parasites that have not yet entered cells and incubate »14-16 h at 37^oC. [Note: The effects of thymidine are parasite density-dependent. It is critical to maintain the multiplicity of this parasite/monolayer/thymidine ratio when scaling this procedure to use more (or less) parasites.] 2.2 Following overnight growth, replace the media with that containing thymidine (1% DMEM, FBS, P/S, AmphB, 10 mM thymidine [Sigma, T1895]). To arrest this population at the G1-S boundary, incubate 4 h at 37^oC. Parasites can be harvested at this time for analysis of the growth-arrested population or EtOH fixation (see below). 2.3 To release ‘blocked’ tachyzoites and allow for continued (synchronous) growth, wash the monolayer 2X with standard growth media and incubate at 37^oC. This population will transit S and G2+M and return to the G1-S boundary in » 6 h. However, synchronous growth is gradually lost as the population enters the subsequent cell cycle. [Note: The morphology of a fraction (20%) of these parasites will be distorted as a result of the mutagenic properties of thymidine.] 3.0 Ethanol Fixation. 3.1 Pellet filter-purified tachyzoites and resuspend in 300 ml 1X PBS on ice. Fix by the drop-wise addition of ice-cold absolute ethanol (700 ml) with constant shaking. Optimal staining requires fixation for 12 h at –20^oC, but tachyzoites in 70% ethanol can be stored for > 1 month at this temperature without effect on subsequent fluoremetric analyses.
Ó2001 Michael W. White-Department of Veterinary Molecular Biology || questions@montana.edu