Hi everybody.
My question today is about generating free malaria parasite. I would like to know if someone is able to give me a protocol to extract this parasite from RBC,
using saponine ?
using tween (80 or 20) ?
using sorbitol ?
with concentration of ?
and without ultra-centrifugation if it is possible
ps: I have already read MMR 2013 by D. Mattei (chapter 4) so how do you replace Plasmion ?
Thank you
Best Regards
Article Isolation of invasive Plasmodium yoelii merozoites with a lo...
Joe Kimanthi's Article may shed some light on your question if I understood your question correctly.
cc.
Hi,
I agree with you. My purpose is to extract the parasite from the red blood cells. This is my main objective. I hope to find a protocol based on lysis of red blood cells without damaging the parasite.
Best Regards
I have no tried this approach, but it might be helpful:
"Infected human erythrocytes of 20% ring-stage parasitemia were prepared from in vitro culture of clone 7G8 from Brazil [21] by two cycles of sorbitol lysis [22] of a large-scale asynchronous culture."
Lindsey J. Panton, et al. Purification and partial characterization of an unusual protein of Plasmodium falciparum: histidine-rich protein II. Molecular and Biochemical Parasitology, 35 (1989) 149-160.
Also, Pishever discusses infected erythrocyte lysis: US6384015 Pishevar Lysis infected RBCs. Remember you will need to get permission to use any technology that is covered in the Claims of a patent.
Good luck
Synchronisation: classical double sorbitol or sortiol + Percoll (instead of Plasmion).
RBC lysis: 0.1% saponine (w/v) in PBS, 5 min, on ice (the % of saponine is depending upon the "quality" of the saponine, test it before).
Synchronisation: classical double sorbitol or sortiol + Percoll (instead of Plasmion). RBC lysis: 0.1% saponine (w/v) in PBS, 5 min, on ice (the % of saponine is depending upon the "quality" of the saponine, test it before).
thank you all.
My solution is nearly the same that of Philippe Grellier by reading (also) Riley et al 2011 and Jingyang Chen et al 2009. All my solutions are cold (+2°C) . I wash twice the pellet of infected Red Blood Cells (iRBCs) with NaCl. I empty the supernatant and I use saponine 0.2% with vortex or I go back and forth with the pipette.
I incubate the solution 5 min on ice. I empty the supernatant, clean my pellet with NaCl. According to the following analysis (Hrmas, RMN, molecular biology etc) I resupend it (or not) with the good solution/ solvent.
According to me, all steps to wash pellet and incubate are critical.
Best Regards
ps: i try to join a picture
Jérôme Dormoi were you able to obtain viable parasites through this methodology? I tried saponin lysis a few times but I couldn't get viable parasites. maybe i left too much time?
Pedro Rodrigues
Hi, really sorry for the delay in my answer. We do not obtain "viable" parasite because we lost digestive vacuola with the method previously disccused and Plasmodium is an intracellular parasite. Meanwhile if we talk about membrane integrity, Plasmodium is viable - i would try to join a picture from microscopy
Pedro Rodrigues
Saponin is a good detergent, if you incubate your mix (plasmodium+saponin) at room temperature, at 0.3 or even 0.1% of saponin you don not exceed 5 min. It depends also on what you are working on (memebrane, proteine, enzymatic activity or nucleic acid) - I work at 5°C, with ice
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