0.2 cm pulse cuvette

T. brucei 29–13 procyclic forms [21 (link)] were cultured to mid-log phase (0.5–0.8 × 10⁷ cells/ml) in SDM-79 (BioConcept, Allschwil, Switzerland) supplemented with 10% (v/v) fetal bovine serum (FBS, LuBioScience GmbH, Lucerne, Switzerland) at 27°C. Parasites (4–5 × 10⁷) were harvested by centrifugation at 1250 × g for 10 min, washed once in transfection buffer (132 mM NaCl, 8 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.5 mM magnesium acetate, 0.09 mM calcium acetate, pH 7.0), resuspended in 450 μl of the same buffer, and mixed with 15 μg of linearized plasmid. Electroporation was performed with a BTX Electroporation 600 System (Axon Lab, Baden, Switzerland) with one pulse (1.5 kV charging voltage, 2.5 kV resistance, 25 mF capacitance timing, and 186 ohm resistance timing) using a 0.2-cm pulse cuvette (Bio-Rad Laboratories AG, Cressier, Switzerland). Electroporated cells were immediately inoculated in 10 ml SDM-79 containing 10% heat-inactivated FBS. Clones were obtained by limited dilution in 24-well plates in SDM-79, containing 20% conditioned medium, in the presence of 2 μg/ml puromycin (Invitrogen, Carlsbad, CA, USA) for selection. Antibiotic-resistant clones were tested for the presence of the introduced constructs by PCR. RNA interference and expression of HA-tagged constructs was induced by addition of 1 μg/ml tetracycline to parasite cultures.

Parasites [4×10⁷ cells at mid-log growth phase (0.5–0.8×10⁷ cells/ml)] cells were harvested by centrifugation at 1250×g for 10 min, washed once in buffer (132 mM NaCl, 8 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.5 mM magnesium acetate, 0.09 mM calcium acetate, pH 7.0), resuspended in 450 μl of the same buffer, and mixed with 15 μg of linearized plasmid. Electroporation was performed with a BTX Electroporation 600 System (Axon Lab, Baden, Switzerland) with one pulse (1.5 kV charging voltage, 2.5 kV resistance, 25 mF capacitance timing, and 186 Ω resistance timing) using a 0.2-cm pulse cuvette (Bio-Rad). Electroporated cells were immediately inoculated in 10 ml of SDM-79 containing 10% heat-inactivated FBS. Clones were obtained by limited dilution in 24-well plates in SDM-79, containing 20% conditioned medium, in the presence of 2 μg/ml puromycin for selection. Antibiotic-resistant clones were tested by PCR. RNAi was induced by addition of 1 μg/ml tetracycline to parasite cultures. Northern blot analysis was performed as previously described (Serricchio and Bütikofer, 2012 (link)) using a probe generated with primers described above.

T. brucei procyclic and bloodstream forms were harvested at mid-log phase, washed once in buffer (132 mM NaCl, 8 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.5 mM magnesium acetate, 0.09 mM calcium acetate, pH 7.0) and resuspended in fresh buffer at a density of 8 × 10⁷ cells/ml. Subsequently, 440 μl of parasite suspension were mixed with 10–15 μg of digested plasmids and transferred to a 0.2-cm pulse cuvette (Bio-Rad). Electroporation was conducted with a BTX Electroporation 600 system (Axon Lab, Baden, Switzerland) with one pulse (1.5 kV charging voltage, 2.5 kV resistance, 25 microfarads capacitance timing, and 186 resistance timing). Cells were immediately inoculated in 10 ml of procyclic or bloodstream form medium. Dilutions were plated into 24-well plates and after 24 h selected for antibiotic resistance. Clones were obtained by limiting dilution.