I am interested in infecting hepatocyte cell lines with P. berghei-GFP sporozoites.
After infecting the hepatocytes, I will determine the infectivity by quantifying the GFP-positive hepatocytes by flow cytometry.
I am looking for suggestions/protocols, to increase the infectivity.
What is the best cell line? The best P. berghei strain? The best multiplicity of infection? The best infection media? Do you use cytokine treatments?
According to your experience, can you recommend the best protocol to achieve the highest infectivity?
What was the highest infectivity you obtained with your protocol?
Use the HepaRG cells, that cell line give rise to adult human hepatocytes, they keep their phenotype during 3+ weeks after differentiation, ie much longer than the primary and that is key to maximise your infection level and to follow the cycle of the parasite . To my knowledge, the Heparg hepatocytes are infectible with P.Berghei, but also falciparum and probably vivax too from the rumors!
Hi Paulo,
Check Maria Mota's lab papers, most of their work is done by infecting hepatoma cell lines and primary mouse hepatocytes with P. berghei spz, and they were the ones who published the application of flow cytometry to quantify infection and parasite development using GFP-expressing P. berghei spz (what you want to do). Here is the paper http://www.ncbi.nlm.nih.gov/pubmed/17697130.
The most common cell line used to be Huh7 cells (human hepatoma cell line, P. berghei is able to infect human hepatocytes but not red blood cells), and RPMI media supplemented with FBS (which I think it is what's in the paper). In some cases Hepa 1-6 cells could also be used, and these are C57L/J mice hepatoma cells. These were grown and infected using DMEM media supplemented with FBS. All this is published somewhere.
I would check more recent papers just to check whether they've changed the amount of parasites used in recent years, to improve infection.
Hope this helps!
Dear Catarina,
Thank your for your comments.
I am aware of Maria´s work and I know very well the paper you mentioned.
My inquiry in this forum was intended to find new suggestions, new protocols and new ideas to improve the infectivity of P. berghei sporozoites in hepatocytes.
Anyway, thank you for you reply.
Paulo
Hi Paulo,
No prob, I thought you knew but just ref it just in case. Hope the forum helps, is usually very good.
See you,
Catarina
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