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Purification protocolsTissue Culture Media:Frank Hinks modification of Grace's Medium, bought in powder and mixed in the lab, Sigma's TNM-FH. Mixing the media:
Fetal Bovine Serum (5%) was added as well as 50 mg/ml gentomycin and the media was filter sterilized using a Corning Sterile Filter Set with a 0.45 µm pore size. Transferring Cultures: Cultures were kept in Corning Costar Tissue Culture Flasks. In transferring the cultures 5 ml of media was dispensed into the tissue culture flask using a sterile serological pipette. Then a small amount of culture was added to the flask using a sterile pipette, and also a small amount was placed on a slide to check for contamination. Cultures were transferred twice a week. Purifying In Vitro Cell Cultures: In order to separate vegetative cells from pellicles a Percoll gradient solution was used. To make the gradient a gradient maker and a range of concentration from 10-50% was used. The Percoll must be prepared with a final concentration of 10% 1.5M NaCl. The volume of the gradients is determined by how much sample there is. The volume of the gradients were 6 ml and this was able to support sample loads of up to 3x108 cells in 1 ml. The gradient is then centrifuged in a swinging bucket rotor at around 700-1000g for 20 minutes. Pellicles are collected using a syringe, and usually occurred 1-2 ml down from the top of the gradient after centrifugation. This Percoll solution was then diluted down at least 1:5 and spun at 1000g for 15 minutes. This wash step was repeated three times to remove the Percoll. Separating Cysts from In Vivo blood samples or In Vivo preparations: Ludox is best for separating cysts from vegetative cells, or from insect matter. In order to make this gradient the Ludox was diluted into a 10% solution and a 75% solution, which was the range for the gradient. A gradient maker was used to actually form the gradient, and the samples were centrifuged in a swinging bucket rotor for however long it takes to get clear bands (depending on sample usually 90-120 min). Then the bands were harvested using a syringe and the sample was washed 5 times in plenty of water. At least a 1:10 dilution of Ludox to water was typically used. |