THE UNIVERSITY OF CALIFORNIA AT RIVERSIDE *Standard Operating Procedure* *K. Le Roch Laboratory*
TITLE: COMPLETE MEDIUM FOR PARASITE CULTURE		Page 1 of 2
SOP #: KLR1	REVISION LEVEL:	DATE:

1 MATERIALS

Important note: All solutions must be filtered using a 0.22 mm filter before being used in culture. Solutions must stay sterile, use hood!

1.1 500 mL Bottle RPMI Medium 1640 (w/25 mM HEPES and L-glutamine) (Gibco)

1.2 10 mg/mL Gentamicine (Gibco)

1.3 1.36 g/L Hypoxantine (Sigma) Make fresh every time!

Add 0.013 g to 10 mL RPMI;
Dissolve on hot-plate with gentle heat and stirring.

1.4 1M HEPES (Sigma)

1.5 7.5% Sodium Bicarbonate (Gibco)

1.6 20% Glucose (Sigma)

1.7 1M NaOH (Sigma)

1.8 20%Albumax (Gibco)

· Add 25 g to 125 mL RPMI in beaker with stir bar;

· Dissolve by gentle stirring and heating @ 37°C for at least 30 min (until solution is yellow-brown);

· Filter solution with 0.22mm filter;

· Aliquot to 15 mL sterile tubes in 6.25 mL volumes;

· Store @ -20°C.

1.9 Human serum

Incubate @ 56°C for 1 h;
Filter with 0.45 mm filter, then with 0.22 mm filter;
Aliquot to 50 mL sterile tudes in 25 mL volumes;
Store @ -20°C.

1.10 For preparation of gametocyte culture medium only… N-Acetyl Glucosamine (Sigma).

2 PROCEDURE

To a 500 mL bottle of RPMI Medium 1640 add…

2.1 3 mL Gentamicine (10 mg/mL Stock). Final concentration: 0.05 mg/mL

2.2 6 mL Hypoxantine (1.36 mg/mL Fresh Stock). Final concentration 0.014 mg/mL

2.3 22.5 mL HEPES (1M Stock). Final concentration 38.4 mM

2.4 15 mL Sodium Bicarbonate (7.5% Stock). Final concentration 0.20%

2.5 6 mL Glucose (20% Stock). Final concentration 0.20%

2.6 2 mL NaOH (1M Stock). Final concentration 3.4 mM

2.7 25 mL Human serum

2.8 6.25 mL 20% Albumax

2.9 For preparation of gametocyte culture medium only… Add 6.48 g N-Acetyl glucosamine. Final concentration 50 mM. Incubate at 37°C for 1 h to dissolve N-acetyl glucosamine and then filter bottle using 0.22 mm filter.