Cell culture methods

2.2.1.1 Preparation of growth medium for P. falciparum culture

The complete growth medium consists of 500 mL RPMI (Roswell Park Memorial Institute) -1640 medium supplemented with 37.5 mM HEPES buffer solution, 5 mM sodium hydroxide solution, filter sterilised (0.5 μM) 10 mM D-glucose, 2 mM L-Glutamine, , 100 μM hypoxanthine solution, 25 mg/mL gentamicin sulfate, 5% for both human serum and albumax-II.

Incomplete growth medium was prepared the same way as the complete medium, but without 5% of albumax-II or human serum.

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2.2.1.2 Preparation of normal human erythrocytes

NBTS UK supplied fresh human red blood cells type-O-Rhesus positive (ORh+). Human blood was aliquoted in to a 50 mL tube and stored at 4˚C for 2-3 weeks. To prepare 50% haematocrit blood cell (RBC) solution for cell culture, a 50 mL aliquot was centrifuged at 1160 g at room temperature (RT) for 10 minutes. The upper serum phase was removed and an equal volume of incomplete growth medium was added to the pelleted RBCs. The RBCs were re-suspended and then pelleted by centrifugation at 850 g RT for 5 minutes. The process of washing the RBC pellet was repeated twice more as describe above to ensure the complete removal of serum, preservatives and white blood cells (WBCs). To complete the process, an equal volume of incomplete growth medium was added to ensure there was an equal volume of blood and supernatant in the tube. The RBCs at 50% haematocrit (HCT) were stored for up to 10 at 4C°.

2.2.1.3 In vitro intraerythrocytic culture of P. falciparum

Two clones of P. falciparum were used in this study; Pf 3D7 is derived from P. falciparum NF54 isolated from a Dutch malaria patient, which is chloroquine sensitive (Delemarre and Van der Kaay, 1979; Walliker et al., 1987), and Pf Dd2Luc _{transgenic parasite line is a clone}

derived from genetic modification of AHE1 (Hasenkamp et al., 2013). Which has a

pfpcna/luciferase expression cassette introduced (Wong et al., 2011), and is chloroquine

resistant.

The P. falciparum strain 3D7 and Dd2Luc were continuously cultured at a 2% HCT and 2% parasitemia as previously described (Trager and Jensen, 1976; Freese et al., 1988). Cultures are maintained at 37°C in an atmosphere of 1% O2, 3% carbon dioxide and 96% nitrogen.

Light microscopy was used to assess the growth and stages of the parasite. Parasite density was controlled by diluting cultures with complete medium, RBC and infected red blood cells

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(iRBCs) as necessary to between 2-4% HCT and 0.5-5 % parasitaemia (PCT) depending on the requirements of the assays.

2.2.1.4 Assessment of Parasitaemia with Giemsa Staining

Parasite density was assessed daily. Thin blood smears were prepared on glass slides and fixed with 100% absolute methanol for 1 minute. The slide was air dried, then covered with 10% giemsa stain (filtered through a 0.45 μM pore filter) and left for 10 minutes. The dye was washed with water and allowed to dry. Parasitemia and life cycle staging were assessed by light microscopy (oil immersion objective lens) at x1000 magnification (Olympus).

2.2.1.5 P. falciparum culture synchronisation using sorbitol-lysis method

P. falciparum culture synchronisation with sorbitol was originally described by Lambros

and Vanderberg (1979). Cells were grown until the culture displays predominantly ring stage parasites in 0-18 hours post RBC infection. The iRBC cell pellet was collected from the parasite culture by centrifugation at 300 g, RT, RT for 5 minutes. The supernatant was discarded and 5 volumes of pre-warmed 5% w/v sorbitol solution was added to the cell pellet and 5 minutes incubation at 37°C. Synchronied iRBC were collected by centrifugation of the culture at 850 g RT for 5 minutes the supernatant was removed and the cell pellet (iRBC represents early ring stage parasites). The culture was put in a flask with the appropriate volume of complete medium, gassed and returned to the incubator at 37 ˚C.

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2.2.2 Trypanosoma brucei

2.2.2.1 In vitro culture of T. brucei

The procyclic forms of T. brucei 427 SMWT strain cells were maintained in HMI-9 medium (a stock of HMI-9 was prepared by dissolving 16 g of HMI-9, 1.51 g of sodium bicarbonate (NaHCO3) and 7 µL of 2M β-mercaptoethanol in 400 mL dH2O), supplemented with 10%

(v/v) foetal calf serum, 2 mM L-glutamine (Gibco) and 100 U/mL penicillin (Gibco) and 100 μg/mL streptomycin (Gibco) at 37°C with 5% CO2 (Hirumi and Hirumi, 1989; Sullivan

et al., 2015). Cell cultures were diluted 1:20 into fresh medium every 3 days to maintain the

cell densities between 105 and 106 cells/mL.

2.2.3 Leishmania mexicana

Procyclic L. mexicana promastigotes (strain MNYC/BZ/62/M379) were maintained in Schneider’s medium (Gibco) pH 7.0 with 10% FBS (Fetal Bovine Serum) (Gibco), 100 U/mL penicillin (Lonza) and 100 μg/mL streptomycin (Lonza) at 26°C. Differentiation to axenic amastigotes were performed by a 1 in 10 dilution of stationary phase promastigotes into Schneider’s medium pH 5.5 supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (complete Schneider’s media pH 5.5) at 32°C (Heather et al., 1997). The density of parasite growth was determined by the addition an equal volume of culture and 2% formaldehyde (v/v) in phosphate buffered saline (PBS), and counted in a Neubauer haemocytometer under Light microscopy.

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2.2.3.2 Generation of plasmid constructs and L. mexicana transfection

Transgenic L. mexicana NanoLuc and NanoLuc-PEST provided by Dr. Berry and described in (Berry et al., 2018). Briefly, NanoLuc and NanoLuc-PEST open reading frames were amplified by PCR from plasmid DNA templates: pNL1.1, pNL1.2 (Promega). All oligonucleotide sequences are provided in Table 2.1. Amplified genes were digested with

BamHI and KpnI and ligated into pSSU-No (Oyola et al., 2012) to produce the constructs

pSSU-NanoLuc and pSSU-NanoLuc-PEST, foer constitutive expression in Leishmania

mexicana. The pSSU expression vector contains flanking regions for integration into the

rDNA locus of the parasite genome. The constructs (PacI/MssI digested) were transfected into mid-log L. mexicana procyclic cells by nucleofection using a 4b Nucleofector system (Lonza), as described previously (Burkard et al., 2007). Transformants were selected after 24 hours by the addition of 40 µg/ml Geneticin (Life Technologies). Integration of the construct into the genome was assessed by PCR amplification of 160-200 ng genomic DNA, using the oligonucleotide primers pSSU-F (region of the 18S gene) and pSSU-R (splice acceptor site in the pSSU vector). Genomic DNA was purified from mid-log promastigote cells using the DNeasy Blood and Tissue Kit (Qiagen).

Table 2.1: Oligonucleotide sequences for cloning and integration.

Name Purpose Sequence

NanoLuc-F

Amplification of NLuc and NLucP for pSSU- Neo cloning 5ʹ -GTTGGTGGATCCACCATGGTCTTCACAC-3ʹ NanoLuc-R 5ʹ -GCCCCGGTACCAGAGTCGCGGCCTTACG-3ʹ NanoLuc- PEST-R 5’- GCCCCGGTACCAGAGTCGCGGCCTTAG-3’

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2.2.3.3 Long term storage of promastigote cells culture

500 µl promastigote cells culture was mixed with 500 µl Schneiders medium pH 7 and 10% DMSO in sterile freezing vials. The vials were stored -80°C overnight before storage for long term in liquid nitrogen.

To defrost cells: A vial of frozen promastigote cells were thawed at 37˚C in water bath.The

cells were then transfared to 1.5 mL eppendorf tube and centrifuged for 5 minutes at 300 g to remove the DMSO. The cell pellet was resuspended in 10 mL Schneiders medium pH 7 and incubated at 26°C.