C ALCOGENUROS C LÚSTER T RINUCLEARES DE M OLIBDENO

All procedures were carried out in the pathogen laboratory (University of Salford) sterile hood (ESCO class II Biological safety cabinet), using aseptic techniques and pre-sterilised equipment. Virkon (Antec International, UK) was used to disinfect waste material before autoclaving and disposal. All routine culture methods are consistent with those employed by Read and Hyde (1993).

2.1.1 Preparation of complete mediim

RPMI 1640 1x (+) L-Glutamine (+) 25 mM HEPES (4-(2-Hydroxyethyl)piperazine-1- ethanesulfonic acid) (Gibco, Life Technologies, UK) was used as the basis of the culture medium. Four additives; 2.5 g Albumin bovine serum fraction V (Sigma, UK), 2.5 ml 1 mg/ml hypoxanthine (Sigma, UK) in phosphate buffered saline (PBS) (Fisher Chemical, UK), 2.5 ml 40% glucose (Dextrose Anhydrous, Fisher Scientific, UK) in sterile water and 0.5 ml 50 mg/ml gentamycin (Sigma, UK) in PBS were transferred to a 50 ml falcon tube along with approximately 20 ml RPMI 1640 from a newly opened bottle. The contents of the falcon tube were allowed to dissolve and were subsequently passed through a 0.22 µm filter directly into the 500 ml bottle of RPMI 1640 medium using a 20 ml syringe. Following gentle mixing, the complete medium was stored at 2-8oC.

2.1.2 Washing Medium

A 500 ml bottle of pre-sterilised RPMI 1640 1x (+) L-Glutamine (+) 25mM Hepes (Gibco, Life Technologies, UK ) without additives was used as washing medium throughout the study and stored at 2-8oC for up to 2 weeks.

2.1.3 Preparation of human blood for culture of Plasmodium falciparum

To remove leukocytes, O+ whole blood (obtained from the human blood bank) was washed immediately before use. An equal volume of whole blood was added to two 50 ml falcon tubes and centrifuged for 5 mins and 3,400 rpm. Following centrifugation, blood plasma and the pale layer (the buffy coat-containing white blood cells) that forms on top of the red blood cells was removed. Blood was then re-suspended in an equal volume of washing medium and following centrifugation (as described previously) the supernatant,

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along with the buffy coat, was discarded. The washing process was repeated 3x to ensure that all leukocytes were removed from the blood. For the latter of the three washes, complete medium not washing medium was used. Finally, full blood (100% haematocrit) was re-suspended in an equal volume of complete medium to give a final haematocrit of 50% and stored at 2-8oC until further use.

2.1.4. In vitro Plasmodium falciparum culture

To begin a new culture, 10 ml of complete medium and 0.5 ml of washed blood was added to a 50 ml culture flask and warmed to 37oC prior to the addition of the parasites. Approximately 0.5 ml of parasitised blood (retrieved from liquid nitrogen, refer to section 2.1.7) was then added to the warmed culture medium to give a final haematocrit of 5 %. Following inoculation, the parasite culture was gassed with a 5 % CO2, 5 % O2 and 90 % N2 gas mixture (BOC Limited, UK) and placed in the incubator (Leec culture safe touch 190 CO2, Leec Limited, Uk) at 37oC.

2.1.5 Routine maintenance of a Plasmodium falciparum culture

Parasites were routinely cultured at a final volume of either 10 ml or 30 ml in 25 cm3 or 75 cm3 flasks respectively. For experimental set up, smaller flasks (12.5 cm3) with a final culture volume of 5 ml were used. For each of the varying flasks volumes, the culture was maintained at approximately 5 % final haematocrit (for every 10 ml of complete medium 1 ml of 50 % haematocrit blood was added). The medium was changed at either 48 or 72 hour intervals. In brief, spent medium was removed and discarded without dislodging the parasitised blood layer that formed at the bottom of the flask. Subsequent to the estimation of parasitaemia (see section 2.1.6), the continuous culture was diluted to 0.5-1 % parasitaemia with washed blood (50 % haematocrit). New complete medium was added to give a final haematocrit of 5 %. Both washed blood and complete medium had been warmed prior to the dilution and re-suspension procedure. The culture was then gassed and placed in the incubator under conditions described previously.

2.1.6 Estimation of parasitaemia

One drop of concentrated parasitised blood (from the bottom of a culture flask) was transferred to the outer margin of a microscope slide. The blood was thinly smeared across the slide using a second slide and a single swift smooth action to obtain a monolayer of cells. The slide was then air dried at room temperature and fixed by rinsing in 100%

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methanol. After an additional air drying step, the slide was immersed in Giemsa stain at room temperature for ~20 minutes. The Giemsa staining solution was prepared by diluting Gurr’s Giemsa stain solution (BDH/VWR international limited, UK) 1:10 with Giemsa buffer. To prepare the buffer solution one tablet pH 6.4 (BDH laboratory supplies, England) was added to 1 litre of freshly distilled water. Following staining, the slides were rinsed with a gentle stream of tap water, dabbed dry and viewed under oil immersion (x 100) using a Leica DM 500 compound microscope. Parasitaemia was estimated by counting the total number of red blood cells per field of view (approx 100-200) and noting those containing parasites. Multiple infections were counted as one (as only one parasite is expected to reach full development). For each slide, at least 3 fields of view were counted from which the average percentage of infected cells was calculated.

2.1.7 Preservation in liquid nitrogen

A predominantly ring stage culture at approximately 15-20 % parasitaemia was selected for preservation in liquid nitrogen. In brief, the culture was centrifuged at 3,400 rpm for 5 min, the supernatant removed and the culture reconstituted to a 50 % haematocrit by adding an equal volume of warmed complete medium. Aliquots (0.5 ml) of the suspension were transferred into 2 ml cryotubes and 0.5 ml 20 % dimethyl sulphoxide (sterile filtered DMSO, Sigma, UK) in Ringer’s solution (9 g NaCl, 0.42 g KCl and 0.25 g CaCl2/ Litre) was added. The tubes were immediately snap-frozen in liquid nitrogen for preservation and storage.

2.1.8 Retrieval from liquid nitrogen

Following an initial thawing period at 37oC the contents of the cryotube were transferred to a microcentrifuge tube and centrifuged at 14,000 rpm for ~1 min using the minispin (eppendorf, UK) centrifuge. Once the supernatant had been discarded the parasitised blood was gently re-suspended in 1 ml of 10% sorbitol (Fisher Scientific, UK) solution (in PBS) with continuous mixing. This was then centrifuged and the process repeated twice more with subsequent re-suspensions in 5 % sorbitol (in PBS) and finally, complete medium. Following the latter washing step in complete medium, the culture was re- suspended in complete medium and inoculated into a culture flask containing 0.5 ml of newly washed blood (50 % haematocrit) and 10 ml of complete medium (see 2.14).

32 2.1.9 Sorbitol synchronisation

During continuous culture P. falciparum parasites rapidly loose synchronisation. If synchronisation was required prior to experimental set-up, sorbitol synchronisation was used to obtain a predominantly ring stage parasite culture. Sorbitol (5 % w/v) was prepared in distilled water and filtered through a 0.22 µm filter. The sorbitol solution was then added directly to pelleted parasite culture (9 ml to 1 ml of culture pellet) and incubated for 5 mins at room temperature. Following this, the culture was centrifuged at 3,400 rpm for 5 mins and the supernatant was discarded. The parasite pellet was then subjected to three washing steps in complete medium, before re-suspension in complete medium at 50 % haematocrit. The synchronised parasite culture was used to set up a new culture as described previously (section 2.1.4).