I want to do some in vitro infections of dendritic cells with Leishmania parasites. In protocols I have seen , parasites are co-cultured with DCs for 4 h and then those that have not entered the cells need to be washed away by 2-3 centrifugations in order to study parasite's transformation into amastigotes and their multiplication . However, since the above cultures are in suspension, parasites are not washed away. Can anyone suggest a solution for this?
Thank you all in advance.
Sedimentation of Leishmania parasite needs a greater centrifugal force (2000 g to 3000 g) compared to the cells (100 g to 600 g). So I would suggest you standardize the centrifugation in the range of 100 to 600 g (your aim should be avoiding the loss of DC). I assume more than 90% of free parasitic cells should be washed off with 3x centrifugation at this speed. For more details refer the following article which describes the isolation of Leishmania from spleens involving separation of cells from Leishmania. Make sure that you use V-bottom tubes for compact pellet formation for DC. I hope this information helps. Good luck
Article Refinement of techniques for the propagation of Leishmania d...
Thank you very much for your answer. I have tried 2-3 different speeds of centrifuge, but unfortunately only a small number of parasites were washed and when I tried lower speed I actually lost a number of DCs.
Then, if you are really keen to get rid off the parasite, I suggest to use gradient centrifugation using percol as suggested in the previous answer's reference. Good luck
Hello, you can use treated plates for adherence, therefore your cells will be attached to the plate and you can wash the parasites only by pipetting. If you wash cells in suspension there are going to be some remaining parasites but you don't need to be aware as they will be phagocytosed anyway. I hope you already sorted otherwise let me know and I can send you some protocols. Regards ;)
Hello, thank you very much for your answer. I have tried poly L lysine treated lamelles. Can you suggest me maybe another adherence factor? Kind regards;)
DCs are highly adherent in nature. If you are culturing them in treated culture plate then adherence is increased very much and its tough to remove from plate. Martinez suggested you rightly to just pippet off to remove excess parasites and the remaing will be phagocytosed by the cells. I hope you have already sorted the issue with her suggestion.
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