I am isolating Plasmodium falciparum merozoite from 3D7 culture, I am doing sorbitol synchronisation and enrichment through MACS column, followed by maturation of parasite in mature schizont, Than I am passing mature schizont through 1.2 um syringe filter. Every steps are going fine, I am able to get merozoite at the end but it is in ~1 ml volume which is higher enough I want to concentrate in 50-100 ul volume. I tried multiple time to do this by spinning at different speed (up to 10000 rpm) but not successful and I lost larger proportion of merozoite by this process. Can any one share their protocols, even if it is different method. Or if there is optimised protocol that can be used to concentrate merozoite.
Hi Surendra, what do you want to do with the meros? If you just want to use them for protein extraction or something like that I would spin them at MAX speed in a microcentrifuge. I don't recall having problems pelleting them at this speed. If you want invasion competent meros then you can't spin too fast. I've often used less than 1ml (down to 500ul) though of course you will get more meros trapped in the filter but the meros that go through will be more concentrated.
Cheers
Dave
Hi surender you can percipitate merozoite using PEG . first you can standardize right percentage of PEG.
Thanks Dave !!
Yes, I am able to get merozoite in sufficient amount after spinning at higher speed (3000 g). I agree that for invasion, high speed centrifugation will be not good idea.
Dear Madam,
Thank for your suggestion about PEG protocol. Is there any paper you may refer that uses PEG? I want to know if this protocol could be more productive.
Hi Surendra, we normally use plasmagel floated schizonts and reincubate them a couple of ours (judge from the floated material) without any new erys and then spin this material down. Until now, we only used this for IFA or protein extraction purposes.
Thanhs Gerhard,
I m doing quite similar way, making merozoite from naturally ruptured schizonts. Only problem now is purity of merozoites. We are getting higher amount of host cell debris which interfere our IFA.
Hi,
Our merozoite purification protocol is published in the open access publication "Methods in Malaria Research' at MR4
See http://www.mr4.org/Publications/MethodsinMalariaResearch.aspx
We also published a detail protocol here:
http://www.pnas.org/content/107/32/14378.long
There is a protocol in the supplementary material
We generally avoid high speed centrifugation
I hope these help
Cheers
James
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