Adapted from Overath et al  (1986) European Journal of Biochemistry 160:175

Materials

• 10xDTM (store in 50 ml aliquots at -20°C) – see bottom of page for recipe
• Hemin (Sigma; 2.5 mg/ml in 50 mM NaOH; sterile filter and store at 4°C)
• 100x CCA (300 mM sodium citrate, 300 mM cis-aconitate; sterile filter and store at -20°C)

Protocol

• Prepare 500 ml DTM as follows and store at 4°C for up to 3 months

	grams unless stated
10xDTM	50 ml
NaCl	3.4
Hepes	3.75
Vitamin solution (Life Technologies)	5 ml
Bring to 400 ml and pH to 7.2 with NaOH
NaHCO3	1.1
Pen-Strep (Life Technologies)	5 ml
Glutamic acid	0.12
L-Glutamine	0.67
Glycerol	0.38 ml
Proline	0.32
Hemin	1.5 ml
2-β-mercaptoethanol	7 μl
Bring to 425 ml, sterile filter and add 75 ml heat-inactivated FBS (Sigma)

• Grow bloodstream form T. brucei to ~2×10⁶ cells per ml in a 10 ml culture volume
• Pellet the culture at 1000 g for 10 minutes and resuspend in 5 ml DTM/CCA, giving a final cell density of 4×10⁶ per ml
• Place the culture in a sealed flask at 27°C
• Over the first 24 hours the cell population remains static, subsequently the cells will typically double approximately every 20 hours (MITat 1.2, clone 221a); therefore, cultures are normally diltuted daily 1:1 in DTM/CCA
• Differentiated cells are normally stable for 10 to 14 days, after which the culture collapses irrevocably

NB: Unlike bloodstream form T. brucei, procyclics are sensitive to growth at low density, and differentiated cells need to be maintained at higher than 1×10⁶ cells per ml – they can be grown to at least 1×10⁷ cells per ml.

 

10xDTM	grams per litre
KCl	4
CaCl2	2
NaH2PO4.H2O	1.4
MgSO4.7H2O	2
Sodium pyruvate	1.1
Phenol red	0.1
Hypoxanthine	0.14
Alanine	0.09
Arginine.HCl	1.26
Asparagine	0.15
Aspartic acid	0.14
Cysteine	0.24
Glutamic acid	0.15
L-Glutamine	3
Glycine	0.08
Histidine.HCl.H2O	0.42
Isoleucine	0.52
Leucine	0.52
LycinexHCl	0.73
Methionine	0.15
Phenylalanine	0.32
Proline	0.06
Serine	0.11
Threonine	0.48
Tryptophan	0.1
Tyrosine	0.36
Valine	0.46