Procyclic-form culture

SDM-79 powder is custom made by Life Technologies and is generally purchased on a consortium basis

One pot is sufficient to make up 5 litres after addition of the following:

Sodium bicarbonate 10 g
Pen-Strep (Life Technologies) 50 ml

Adjust to pH 7.4; make up to 4.5 litres with distilled water, filter sterilise, store at 4°C in 450 ml volumes.

Prior to use, add 50 ml heat-inactivated fetal bovine serum (FBS; Sigma) and 1.5 ml hemin* (Sigma); store at 4°C for up to two months.

*Hemin stock solution – 2.5 mg/ml in 50 mM NaOH; filter sterilise and store as 20 ml aliquots at 4°C.

Cultures should be grown in non-vented flasks at 28°C, and maintained at a density of 1×106 to 2×107 cells per ml.

NB: growth at low density should be avoided unless in the presence of conditioned medium (grow a culture to ~1×107 cells per ml centrifuge twice and filter sterilise through a 0.2 µm filter to remove all cells, and supplement with 10% heat-inactivated FBS; use within one week) or wild type cells under drug selection.