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Purification of Toxoplasma gondii from cell cultures (Part A)Separation of sporocysts and oocyst walls (Part B) ♦ OptiPrep™ is a 60% (w/v) solution of iodixanol in water, density = 1.32 g/ml♦ To access other Application Sheets referred to in the text return to the Cell Index; key Ctrl “F” andtype the C-Number in the Find Box Part A 1. BackgroundToxoplasma gondii can be maintained in cell culture using Vero, Chinese hamster ovary (CHO)cells or human foreskin fibroblasts (HFF). Coppens et al [1] developed a simple continuous 10-30%Nycodenz® gradient to purify the parasite cells away from the host cell material; it has been used inmany later studies [2-6]. Although iodixanol gradients have been used separating oocyst walls andsporocysts, they have not been used in this particular Toxoplasma application. Since Nycodenz® isonly available as a powder, while iodixanol solutions are prepared by simple dilution of OptiPrep™,this alternative is given in the methodology below. Although it is highly likely that this modificationwould be effective in the purification of the organism, it has not been validated.The following methodology is adapted from ref 1.2. Solution preparationA. Nycodenz® powder ORB. OptiPrep™ (shake gently before use)C. Phosphate-buffered saline (PBS)D. For Nycodenz® solutions only: Phosphate buffer: 100 vol. of 1.78% (w/v) Na2HPO4.2H2O + 25vol. 1.38% (w/v) NaH2PO4.H2OTo make up a 30% (w/v) Nycodenz® stock solution place 50 ml of water in a 150 ml beaker on aheated magnetic stirrer set at approx. 50°C and add 30 g of Nycodenz® powder in small amounts untildissolved. Allow the solution to cool to room temperature; add 5 ml of Solution D and then make up to100 ml with water. Filter sterilize if required (see Note 1). For the continuous gradient prepare also asolution of 10% (w/v) Nycodenz® by diluting the 30% stock solution with Solution C. For adiscontinuous gradient alternative also make up similarly a 20% Nycodenz® solution (see Section 3,Step 2).For the iodixanol option simply dilute OptiPrep™ (60% w/v iodixanol) with Solution C to make a10% and 30% OR 10%, 20% and 30% (w/v) iodixanol solutions (see Note 1 and Section 3, Step 2).3. Protocol1. Culture the Toxoplasma in Vero cells, CHO cells or HFF (see ref 1).2. Preparation prepare continuous gradients (total volume approx 8 ml in a 15 ml tube or approx 30ml in a 50 ml tube) of Nycodenz® or iodixanol from equal volumes of 10% and 30% Nycodenz®or iodixanol using a two-chamber gradient maker or Gradient Master™. If neither of these devicesis available prepare discontinuous gradients from equal volumes of 10%, 20% and 30%Nycodenz® or iodixanol; carefully rotate the tubes to a horizontal position and allow the gradientto form by diffusion (see Note 2).3. During gradient production harvest parasites from the cell culture supernatants and pass thesuspensions twice through a 27-gauge syringe needle to disrupt any contaminating cells. Excellence in Separations OptiPrep™ Application Sheet C33

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2 4. Wash the Toxoplasma three times in Solution C; centrifuging the suspension each time at 1000 gfor 10 min.5. Finally suspend the pellet in 10% Nycodenz® or iodixanol and layer on top of the continuousgradient (see Note 3).6. Centrifuge at 2000 g for approx. 30 min (see Note 4).7. Harvest the Toxoplasma that bands around 1.09-1.11 g/ml (just above half way down the gradient).4. Notes1. The 30% (w/v) Nycodenz® solution will be slightly hyperosmotic (approx. 315 mOsm); all theiodixanol solutions will be isoosmotic with mammalian cells.2. Diffusion of the discontinuous gradient should take no more than about 1 h at room temperature. Ifthe tubes are kept vertical, the process will take several hours. For more information see Application Sheet C02. Because the Toxoplasma bands at a density just below that of 20% (w/v) Nycodenz® or iodixanol, a discontinuous gradient may be effective in the purification process butthis has not been tested (as far as we know).3. The residual buffer on and in the pellet will dilute the gradient medium to allow layering on the10-30% gradient. If difficulty is encountered in layering the sample, dilute it with about 0.2 ml ofSolution C.4. Do not use the brake for deceleration of the rotor.5. References 1. Coppens, I., Sinai, A.P. and Joiner, K.A. (2000) Toxoplasma gondii exploits low-density lipoprotein receptor-mediatedendocytosis for cholesterol acquisition J. Cell Biol., 149, 167-180E2. Coppens, I. and Joiner, K.A. (2003) Host but not parasite cholesterol controls Toxoplasma cell entry by modulatingorganelle discharge Mol. Biol. Cell, 14, 3804-38203. Quittnat, F., Nishikawa, Y., Stedman, T.T., Voelker, D.R., Choi, J-Y., Zahn, M.M., Murphy, R.C., Barkley, R.M.,Pypaert, M., Joiner, K.A. and Coppens, I. (2004) On the biogenesis of lipid bodies in ancient eukaryotes: synthesis oftriacylglycerols by a Toxoplasma DGAT1-related enzyme Mol. Biochem. Parasitol., 138, 107-1224. Nishikawa, Y., Quittnat, F., Stedman, T.T., Voelker, D.R., Choi, J-Y., Zahn, M.., Yang, M., Pypaert, M., Joiner, K.A.and Coppens, al (2005) Host cell lipids control cholesteryl ester synthesis and storage in intracellular ToxoplasmaCell. Microbiol., 7, 849-8675. Massimine, K.M., Doan, L.T., Atreya, C.A., Stedman, T.T., Anderson, K.S., Joiner, K.A. and Coppens, I et al (2005)Toxoplasma gondii is capable of exogenous folate transport a likely expansion of the BT1 family of transmembraneproteins Mol. Biochem. Parasitol., 144, 44-546. Sehgal, A., Bettiol, S., Pypaert, M., Wenk, M.R., Kaasch, A., Blader, I.J., Joiner, K.A. and Coppens, I. (2005)Peculiarities of host cholesterol transport to the unique intracellular vacuole containing Toxoplasma Traffic, 6, 1125-1141 Part B 1. BackgroundThe resistance of Toxoplasma gondii is thought to be related to the oocyst wall that surrounds thesporocysts [1]. To investigate the nature and functional properties of the oocyst wall, divorced from thesporocysts could be an important step in understanding the infectious properties of this organism.Although Percoll® gradients were able to provide a purified sporocyst fraction, because theseparticles do not all band in a discrete manner in such gradients, they were unable to provide asimultaneous isolation of a pure oocyst wall fraction [1]. Gradients formed from OptiPrep™ on theother hand are able to provide purified sporocysts and oocyst walls in the same gradient.The following protocol is adapted from ref 1. Everson et al investigated top-loaded and bottom-loaded discontinuous iodixanol gradients and both alternatives are presented in this Application Sheet.

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3 It describes only the gradient separation and not the method for mechanical fragmentation of theoocysts –see ref 1 for this information. See ref 2 for a review of Toxoplasma methodology.2. Solutions requiredE. OptiPrep™ (shake gently before use)F. Diluent: 0.25 M sucrose, 15 mM Tris-HCl, pH 7.53. Protocol1. Fragment the oocysts using glass beads andvortexing according to ref 1.2. Prepare the following density gradient solutions by diluting OptiPrep™ with Solution B: 2.5%, 5%,10%, 15%, 20%, 25% and 30% (v/v) OptiPrep™. For bottom loading omit the 30% OptiPrep™(see Note 1 and Important Note at the end of Section 4).3. For bottom loading only: mix 7 vol. of the fragmented oocyst suspension with 3 vol. of OptiPrep™(i.e. adjust the suspension to 30% v/v OptiPrep™) and use this in place of the 30% OptiPrep™(step 1).4. Prepare a discontinuous gradient from equal volumes (1-2 ml) of each of the iodixanol solutions;Underlayering is probably the easiest way of creating the gradient (see Notes 2-4).5. For top-loading only, load the gradient with the fragmented oocysts in 2.5% v/v OptiPrep™ (seeNote 5).6. Centrifuge at 1000 g for approx. 1 h (see Note 6). Do not use the brake for deceleration.7. Harvest the intact sporocysts which band between 5% and15% iodixanol and the oocyst walls which band at the25%/30% interface (see Figure 1 and Notes 7 and 8).4. Notes1. Everson et al [1] investigated gradients with step intervalsof both 2.5% and 5% OptiPrep™.2. Underlayering using a metal cannula attached to a 1-2 mlsyringe is the best way of creating multiple step gradients.Alternatively a small volume (“low-pulse”) peristalticpump might be used to introduce each layer, dense endfirst. Use the pump to take up the aliquot of solution andthen reverse the flow to expel into the centrifuge tube. Formore information see Application Sheet C02.3. The gradient will become more or less continuous(particularly if 2.5% steps are used) due to the mixingthat is bound to occur and diffusion during the setting upand centrifugation.4. Dumètre and Dardè [3] used bottom loading with slightlydifferent discontinuous iodixanol gradient of 30%, 25%, 20%, 15% and 5% (v/v). This was alsoused by Fritz et al [4].5. Top-loaded gradients tend to give a lower yield of oocyst walls than do bottom-loaded gradients.Although the yield of sporocysts was greater with top-loaded gradients, contamination by somenon-sporulated oocysts was greater.6. Everson et al [1] investigated centrifugation times from 20-100 min. One hour is probably optimal,although if flotation is used some of the non-sporulated oocysts, which are found below the 15%iodixanol, have probably not had time to reach their banding density. Keep the following stock solution at 4°C:1 M Tris (free base), 12.1 g per 100 ml waterSolution F: Dissolve 8.5 g sucrose in 50 ml water; add1.5 ml of Tris stock; adjust to pH 7.5 with 1 M HCland make up to 100 ml. Figure 1: Separation of sporocysts and oocystwalls from fragmented Toxoplasma gondii, in adiscontinuous iodixanol gradient. Sample maybe loaded at either of the two arrowedpositions. 1000g/60 min 12108426 2.5%5.0%10%15%20%25%30% 12108426 SporocystsOocyst walls

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47. Either aspirate observable bands of material or unload the entire gradient by careful aspirationfrom the meniscus; use a flat-tipped metal cannula (0.8 mm i.d.) attached to a 1-2 ml syringe. Mostgradient unloaders are designed for use with flexible thin-walled tubes and not the screw-capthick-walled tubes routinely used for cells. For more information regarding the harvesting ofgradients see Application Sheet S08, accessed from the “Subcellular Membranes” index.8. Other groups have also reported the use of these OptiPrep™ techniques for the studies of oocystsand sporocysts (e.g. see ref 5 and 6).IMPORTANT NOTE: published papers below often describe gradient solutions as “% v/viodixanol”. Since OptiPrep™ is the commercial name for a 60% (w/v) iodixanol solution it hasbeen assumed that the solutions are actually % v/v OptiPrep™.5. References1.Everson, W. V., Ware, M. W., Dubey, J. P. and Lindquist, H. D. L. (2002) Isolation of purified oocyst walls andsporocysts from Toxoplasma gondii J. Eukaryot. Microbiol., 49, 344-3492.Dumètre, A. and Dardé, M-L. (2003) How to detect Toxoplasma gondii oocysts in environmental samples FEMSMicrobiol. Rev., 27, 651-6613.Dumètre, A. and Dardé, M-L. (2005) Immunomagnetic separation of Toxoplasma gondii oocysts using a monoclonalantibody directed against the oocyst wall J. Microbiol. Meth., 61, 209-2174.Fritz, H.M., Bowyer, P.W., Bogyo, M., Conrad, P.A. and Boothroyd, J.C. (2012) Proteomic analysis of fractionatedtoxoplasma oocysts reveals clues to their environmental resistance PLoS One 7: e299555.Gondim, L.F.P., Wolf, A., Vrhovec, M.G., Pantchev, N., Bauer, C., Langenmayer, M.C., Bohne, W. Teifke, J. P. et al(2016) Characterization of an IgG monoclonal antibody targeted to both tissue cyst and sporocyst walls of Toxoplasmagondii Exp. Parasitol., 163, 46-566.Escotte-Binet, S., Da Silva, A.M., Cancès, B., Aubert, D., Dubey, J., La Carbona, S., Villena, I. and Poulle, M-L. (2019)A rapid and sensitive method to detect Toxoplasma gondii oocysts in soil samples Vet. Parasitol., 274: 108904OptiPrepTM Application Sheet C33; 6th edition January 2020