Before commencing this protocol, sorbitol synchronised P. falciparum parasites were cultured to mature pigmented trophozoite stage and ~10% parasitaemia.
Procedure:
A) Flow cytometry
Assays are performed in 96 well plates in a total volume of 50 μL but can be scaled down to 25 μL.
1. Incubate parasites at ~10% parasitaemia and 0.5% haematocrit (in incomplete RPMI or 1X PBS) with 10% (heat-inactivated*) immune or non-immune test serum samples and 25% normal serum (NS) as a source of complement for 1 hr at 37°C (final volume of 50 μL).
*Serum test samples should be heat-inactivated prior to use in this assay by incubating at 56°C for 45 minutes to inactivate host complement proteins. Alternatively, purified antibodies from samples can be used.
2. Centrifuge for 2 minutes at 400 x g and aspirate supernatant. Wash cells twice in 0.5% BSA in 1X PBS.
3. Incubate in 50 μL of appropriate anti-human complement antibody, diluted 1:100 dilution in 0.5% BSA in 1X PBS, for 1 hr at room temperature (RT).
4. Aspirate and wash as in Step 2.
5. Incubate in 50 μL of polyclonal Alexa Fluor 488 conjugated antibodies at 1/500 dilution with 1/1000 ethidium bromide for 1 hr at RT.
6. Aspirate and wash as in Step 2.
7. Resuspend in 200 μL 0.5% BSA in 1X PBS and acquire on flow cytometer with appropriate gating for parasitised red blood cells (pRBCs) and compensation for ethidium bromide and Alexa Fluor 488.
8. Levels of complement deposition on pRBCs are expressed as geometric mean Alexa Fluor 488 fluorescent intensity of ethidium bromide stained pRBC populations. (Subtract the signal from the uninfected RBC population from each sample to account for background fluorescence.)
B) Western Blot
1. Incubate parasites at ~10% parasitaemia and 0.5% haematocrit (in incomplete RPMI or 1X PBS) with 25% NS and 10% immune or non-immune test serum samples for 15 minutes at 37°C (final volume of 50 μL).
2. Spin for 2 minutes at 400 x g and aspirate supernatant. Wash cells twice in 200 μL cold 1X PBS containing protease inhibitors.
3. Aspirate as much of the supernatant as possible. The samples can be frozen for longer term storage after this step.
4. Resuspend in 20 μL* 1X reducing SDS samples buffer (i.e. 5 μL 4X sample buffer, 2 μL DTT and 13 μL 1X PBS).
*Scale up/down appropriately depending on the amount of protein you detect on your gel.
5. Boil at 95°C for 5 minutes.
6. Load 10 μL of each sample on your gel and 5 μL of protein standard on one well. Use a NuPAGE 4-12% Bis-Tris 10-lane gel and 1X MES SDS running buffer at 200 V, 200 mA and 20 W for 40-60 minutes.
7. Transfer onto nitrocellulose membranes.
8. Block membrane in 10% skim milk in 1X PBS overnight at 4°C with shaking/rolling.
9. Wash three times in PBS-Tween 0.05%.
10. Incubate membrane with 5 mL anti-complement antibody at 1/5000 dilution in 5% skim milk in 1X PBS at RT for 1 hr.
11. Wash as in Step 9.
12. Incubate membrane with 5 mL appropriate (species-specific) HRP-conjugated antibody, diluted 1/5000 in 5% skim milk in 1X PBS, at RT for 1 hr.
13. Wash as in Step 9.
14. Add 1 mL of chemiluminescent substrate (Thermo Scientific) to membrane and detect signal by exposing to autoradiography X-ray film (GE Life Sciences) timed accordingly.