CHAPTER 2 MATERIALS AND METHODS
2.4 Parasitological techniques
Rodent malaria parasites including P. yoelii, P. berghei, P. vinckei and P. chabaudi are adapted for laboratory studies. All four rodent malaria parasites infect mice. The study of interaction between the host and the erythrocytic stage of the malaria parasite makes the mouse a very useful host for malaria infection models to study immunological, molecular and biochemical aspects of the parasite (Sanni et al., 2002). There is a degree of variation in virulence in various mouse malaria model species and strains. There are strains of P. yoelii that are lethal in mice whereas some other P. yoelii strains are non-virulent in mice.
This section describes several techniques used in the propagation, isolation and purification of P.
yoelii; a rodent malaria parasite. In this study, the lethal P. yoelii stain 17XL was used. Parasites were propagated in 6 – 8 weeks old male BALB/c mice through intraperitoneal injection of 100 µl of 107 parasitized red blood cells. Mice used in this study were kept in the University Animal housing facility in a temperature range of 19 – 22ºC. Each animal was kept in a single cage with a plastic bottom and steel mesh. Cages, saw dust, water and food were autoclaved prior to use. Food and water were available ad libitum. Parasitemia was monitored daily through thin blood smear made from the tail blood.
2.4.1 Propagation of P. yoelii parasite in BALB/c mice and cryo-preservation 2.4.1.1 Materials
Phosphate buffered saline (PBS), pH 7.2. NaCl (8 g), KCl (0.2 g), Na2HPO4 (1.15 g) and KH2PO4 (0.2 g) were dissolved in 1 L of dH2O.
Cryo-preservative [10% (v/v) glycerol-PBS]. Glycerol (1 ml) was added to 9 ml of PBS. The solution was stirred and autoclaved.
2.4.1.2 Method
Parasites were propagated in male BALB/c mice by intra-peritoneal injection of parasite stabilate (107 parasitized red blood cells). The lower abdomen was cleaned with 70% ethanol and the mouse held upside down for the organs to partially drop. The syringe (insulin syringe, 1 cc) was inserted ~ 0.5 cm into the skin and the stabilate slowly released into the mouse. The syringe was slowly withdrawn to prevent exuding of the stabilate after injection. The mouse was immediately release into its cage and monitored for 5 – 10 min post injection. Parasitemia was monitored with daily thin smear of tail blood. The thin smear was air-dried and fixed in methanol for 1 min at RT and was allowed to dry at RT. The smear was stained with Giemsa stain for 20 – 30 min at RT, washed under slow running water and air-dried and viewed at 1000× magnification (oil immersion). The number of infected RBC was expressed as a percentage of total RBC. The parasites were preserved in 10%-glycerol-PBS at 107 parasitized-RBC (pRBC) per 100 µl of stabilate. The stabilate was immediately stored in liquid nitrogen.
2.4.2 Isolation of P. yoelii parasites from infected mouse red blood cells 2.4.2.1 Materials
Phosphate buffered saline (PBS), pH 7.2. NaCl (8 g), KCl (0.2 g), Na2HPO4 (1.15 g) and KH2PO4 (0.2 g) were dissolved in 1 L of dH2O.
Saponin-PBS [0.05% (w/v) saponin in PBS]. Saponin (5 mg) is dissolved in 10 ml of PBS. This reagent is kept on ice prior to use.
Plasmodipur®
Heparinzed vacuum tubes
2.4.2.2 Method
At 30 – 40% parasitemia, the infected mouse was sacrificed. The blood was dispensed into a heparinzed vacuum tube and kept on ice. The blood was diluted (1:4) with ice cold PBS and filtered with Plasmodipur® according to the manufacturer’s instructions to remove host white blood cells. The filtrate was centrifuged (700 g, 15 min at 4ºC) to pellet the infected RBC. The cells were washed 3× with ice cold PBS (5 volumes of the original blood volume) and re- suspended in 5 volumes of saponin-PBS. The cell suspension was incubated at RT for 30 min for complete hemolysis. A dark red colour indicates hemolysis of the RBC. The suspension was centrifuged (3500 g, 15 min at 4ºC) to pellet the released parasites. The supernatant was removed carefully leaving behind a small volume of the supernatant in order not to disturb the parasite pellet. The parasite pellet was washed 2× with 5 volumes of PBS.
2.4.3 Immunofluorescence microscopy 2.4.3.1 Materials
Fixing reagent [4% (w/v) paraformaldehyde-0.0075% (w/v) glutaraldehyde]. Paraformaldehyde (0.4 g) was suspended in 9 ml of ddH2O. Drops of 1M NaOH were added while stirring in a boling water bath. The solution was made up to 10 ml with ddH2O. Glutaraldehyde (1.5 µl of 50% solution) was added. The solution was filtered through a 0.22 µm syringe filter.
Quenching solution (0.1 mg/ml NaCNBH4 in PBS). NaCNBH4 (1.0 mg) was dissolived in 10 ml of PBS. The solution was filtered through a 0.22 µm syringe filter.
Phosphate buffered saline (PBS). NaCl (8 g), KCl (0.2 g), Na2HPO4 (1.15 g) and KH2PO4 (0.2 g) were dissolved in 1 L of dH2O. The solution was filtered through a 0.22 µm syringe filter.
Permeabilizing solution (0.1% Triton X-100 in PBS). Triton X-100 (10 µl) was made up to 10 ml with PBS. The solution was filtered through a 0.22 µm syringe filter.
Saline solution. NaCl, (0.9 g) was made up to 10 ml with dH2O. The solution was sterilized by filtration (0.22 µm syringe filter). The solution was filtered through a 0.22 µm syringe filter.
BSA-PBS solution [3% (w/v) BSA in PBS]. BSA (0.3 g) was dissolved in PBS (10 ml). The solution was filtered through a 0.22 µm syringe filter.
BSA-PBS solution [5% (w/v) BSA in PBS]. BSA (0.5 g) was dissolved in PBS (10 ml). The solution was filtered through a 0.22 µm syringe filter.
DAPI solution [5 µg/ml in PBS]. DAPI (2.5 µl of a 2 mg/ml solution) was made up to 1 ml with PBS. The solution was filtered through a 0.22 µm syringe filter.
2.4.3.2 Method
P. yoelii-infected mouse blood (300 µl) collected in a heparinised-tube was centrifuged (700 g, 10 min, 4ºC), the plasma was discarded and packed cells were resuspended in 1 ml of PBS and washed 5× with PBS (800 g, 10 min, 4ºC) until the supernatant became very clear. The packed cells were resuspended to 2× original blood volume (600 µl) with the Fixing reagent and incubated on ice for 30 min. The cells were washed 5× (800 g, 5 min, 4ºC) with saline solution, resuspended in the quenching reagent (0.5 ml) for 10 min on ice and washed 5× (800 g, 5 min, 4ºC) with saline solution. The cell were incubated in the permibilizing reagent (0.5 ml) for 10 min and washed 3× (800 g, 5 min, RT) with saline solution. The cells were blocked with 0.5 ml of BSA-PBS reagent (1 h, RT); aliquots of 100 µl were transferred to fresh microfuge tubes
and cells were pelleted (800 g, 5 min, RT). The cells were incubated in 5 – 20 µg/ml primary antibody in 3% BSA-PBS (overnight and 4ºC). The cells were equilibrated to room temperature (1 – 2 h), washed 5× (800 g, 5 min, RT) with saline solution and incubated with donkey anti-IgY- FITC secondary antibody (1/50 in 5% BSA-PBS) for 1 h at RT (away from light). The cells were washed 5× (800 g, 5 min, RT) with saline solution, stained with DAPI solution (away from light), washed 5× (800 g, 5 min, RT) with saline solution and resuspended to approximately 50 µl with PBS. A thin smear was made on a microscope slide and cells were allowed to dry overnight in a desiccated vacuum chamber away from light and mounted with SlowFade using coverslip. The edges of the cover slip were sealed with clear nail varnish. There was 5 min incubation period between each wash steps. The cells were viewed with Olympus AX70 fluorescent microscope using a green and UV filters for FITC and DAPI, respectively. Images were captured with CC12 Soft Imaging System™ colour camera and processed with the analySIS™ software.