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.
*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.