BSF to procyclic differentiation

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