Toxoplasma gondii sporozoite excystation from oocysts and purification. Protocol: MJerome060601 Ó2001 Michael White-Veterinary Molecular Biology || Questions at mjerome@montana.edu
WhiteProtocols2001
1.0 Sporozoite excystation from oocysts and purification. ·Oocysts are stored in 2% sulfuric acid at 4^oC. ·Oocysts must be worked with in a laminar flow hood. ·Excysting Media: 0.05 g Trypsin 1.00 g Taurocholic Acid -QS 20 ml in 1X PBS. -stir to mix, pH to 7.5 and sterile filter -warm to 37^oC in a water bath. 2.0 Pellet stored oocysts using a tabletop centrifuge (Sorvall RT6000B at 1300 rpm for 10 min, no brake). Discard supernatant and wash pellet with 10 ml Hanks buffer (pH 7.2) in a 15 ml screw-cap tube. Repeat until sulfuric acid is completely removed (Hanks will turn a bright yellow until the sulfuric acid is gone). 3.0 Add 10 ml 10% Clorox in PBS to sterilize. Let rock for 30 min. Prepare the excysting media. 4.0 Pellet the oocysts for 10 min at 1300 rpm and remove the supernatant. Wash the pellet as described above to remove Chlorox (the media will be purple until washed completely). 5.0 Count the number of total oocysts. If there are more than 30 million oocysts divide them into separate 15 ml tubes. Larger numbers decrease the ability of oocysts to excyst. Spin the tubes down and remove the supernatant. Bring each pellet up in 3 ml of Hanks (pH 7.2). 6.0 Add 4 g of glass beads (Sigma G-9393) and vortex for 45-60 s. Examine for at least 50% of the sporocysts to be free from the oocyst wall. Carefully remove the supernatant using a Pasteur pipette (drawn out if possible) being careful to leave the beads at the bottom of the tube. Wash the glass beads 2 times with 3 ml of Hanks (pH 7.2) and remove the supernatant as described above. Spin 10 min at 1300 rpm to pellet. 7.0 Resuspend the pellet in 5 ml of pre-warmed excysting media. Place the tube with a loosened cap in beaker of water at 37^oC. Stand 30 min in CO2 incubator at 37^oC. Check after 15 min to make sure the sporozoites are excysting. Sporozoites are thin and banana shaped. 8.0 When the sporozoites have excysted, add 5 ml 10% FBS to the media, and centrifuge for 10 min at 1300 rpm. Wash the pellet 2X as above to remove the trypsin. 9.0 Excysted sporozoites are ready to infect a cell monolayer. The sporozoites may be filtered using a 3 mm pore polycarbonate filter to remove oocyst debris, but expect to lose up to half of the sporozoites in the process.
Department of Veterinary Molecular Biology, Montana State University, Bozeman MT