Hello Takano-san,

I have noted a number of things that may help you with your protocol.

First the binding and washing medium is RMPI 1640 adjusted to pH 6.8.

Also, do you enrich your parasites for trophozoites and schizonts by Plasmion? It will help if you do as rings and RBC will not bind so you will need to dilute an enriched culture into binding medium to the required concentration (5x107/ml).

Here are some reference you might find useful as well as the protocol I have used (below).

Pouvelle B et al. (1998). Biological and Biochemical Characteristics of Cytoadhesion of Plasmodium falciparum-Infected Erythrocytes to Chondroitin-4-Sulfate. Infection and Immunity. 66(10):4950-4956.

Buffet PA, et al., Plasmodium falciparum domain mediating adhesion to chondroitin sulfate A: A receptor for human placental infection. PNAS. 96(22): 12743–12748.

I hope this helps.

Good luck and let me know if it works.

Best wishes,

Déla

Materials

Parasites: 3D7 - gelatin-enriched parasites (trophs & schizonts)

Ligands: CD36/SR-B3 Fc Chimera (R&D systems), 1% BSA (Fraction V, Sigma) in PBS.

Binding medium: RMPI 1640 pH 6.8

Procedure:

1. Draw spots (at least in duplicate) ~5mm circle on back of petri dish for the spots. I have attached a diagram for illustration purposes.

1. Immobilization of ligands on petri dish.

    Put 10 ul of 10 ug/mL CD36 recombinant protein and 10 ul of 1% BSA/PBS on the spots.

If you find that 10 ul is drying out or giving you inconsistent results use 20 ul/ spot for all steps instead of 10ul.

2. Incubate the petri O/N at 4C (in humid chamber)

3. Remove the ligand by pipetting or aspiration - be careful not to touch the area of the spot that was coated with the tip - it will remove the ligand from the petri.

4. Block with 10 ul of 1% BSA/PBS for 30 min at RT

5. Remove the BSA by pipetting as in step 3.

6. Put 10 ul of lates stage parasites at  5 x 107/ml on each spot

7. Incubate 60 min at 37 C

8. Wash the dish with 15 mL of  binding medium 5 times- add the medium to the edge of the petri while holding it tilted.

9. To wash tilt the petri dish back and forth, from side to side

10. Tilt the petri dish to aspirate the medium with the pipette (same between each wash)

11. Wash 1  x with PBS (15 ml)

12. Fix 2 hr at RT with 5-10 ml 2% glutaraldehyde

13.Wash 3 x PBS to remove the fixative

14. Stain with 10% Giemsa for 10 min

15. Wash Giemsa with water

16. Air dry the pertri

17. Count under the microscope

I found I needed to add oil to the spots in the dish in order to count properly and double check that I wasn't counting uninfected cells (they should not be binding). Depending on your microscope this may not be necessary but while you are optimising it could be useful. However, if you put immersion oil directly on the spots after a while it will damage them.

Thanks a lot, Ms. Dela-san.

I do not adjust pH of medium to 6.8, but the cultured iRBCs used in assay are usually purified by gelatin sedimentation. Anyhow, it's quite helpful for me! Thanks again!

Did you ever checked your parasites whether they really bind to HUVEC of HMVEC? I am saying this because during long in vitro culture, they may lose their cytoadhesion properties and you need to pan them again to get the adhesive ones.

Hi Hassan,

i have never checked the binding of my parasites to endothelial cells. I have been trying to pan it by using gelatin sedimentation for a while but some could not become an enriched troph/schizont parasites after geratin sedimentation several times. So, my main concern is whether the parasites that have cytoadherence properties can be obtained from such population of parasites by gelatin sedimentation.

By gelatin sedimentation, you can enriched the knoby parasites but still there is controversy between knoby parasites and cytoadhesion. I believe you need to pan them using endothelial cells or ask a cytoadhesive parasite line from other labs.  

Hi Hassan, yes, you are correct.Then, it might be better to pan the parasite lines using endothelial cells. Anyway, thanks a lot!

Takano-san, you can also pan on purified receptors. It is the same principle as for the binding assay. I would need to double check the protocol though for the concentration and amount. I used to do it in T25 flasks. You typically recover very few parasites on the first panning and you will need to do several to enrich for a given binding phenotype.

Regarding what you said about your parasites. You are recovering very few parasites after gelatin flotation? Do you take these trophs and put them back into culture each time?

Hi Dela-san, that's nice. Yes, I performed gelatin flotation three times on 3D7 and a several KO parasites, all of which were generated based on the 3D7 backbone. Only one KO-parasite line was enriched with tropho/schizont-stage of parasites after three round of gelatin flotation, whereas remaining parasite lines including 3D7 could not be enriched. Yes, I always put them back into culture after each gelatin sedimentation. These gelatin-sedimented parasites were grown for 7 days and used for binding assay usually.

Hi Takano-san, do your 3D7 still have HRPII/KAHRP?

OK, then it's top priority for me to see whether or not my 3D7 still retains HRPII/KAHRP. I will check this by RT-PCR from now on. Thanks Dela-san!