I have a stock of RBC which is more than one month old but still my parasites are growing well in them as observed using giemsa staining. I'm planning for stable transfection. So can I use the same RBC (after washing step) or should I use fresh RBC.
Does change in batch of RBC change the growth of parasites.
We always transfect into fresh RBCs (certainly less than 2 weeks from blood draw), even if the parasite look like they're growing ok in older blood. For a transfection, you want your parasites growing optimally and they are generally acknowledged to grow best in freshly-drawn blood because, as Praveen notes, blood cells age over time and parasites grow in the youngest cells most efficiently. We also 'feed' them weekly after transfection for the same reason (cells age even faster at 37C than when stored in the fridge). Because blood batches do differ, Kunal's suggestion to mix a couple of different batches may also help your transfections to succeed.
See reference on optimal transfection procedures:
'A quantitative analysis of Plasmodium falciparum transfection using DNA-loaded erythrocytes.'
Hasenkamp, S., Merrick, C.J., Horrocks, P. Molecular and Biochemical Parasitology 187(2), 117-120 (2013).
Old RBCs become fragile with time and tend to lyse gradually in routine culture itself, which later makes entire culture unhealthy and a probable shift to gametocyte differentiation with time.
For transfections, always use fresh erythrocytes.
change in RBC does change the growth of parasite as you cant predict the surface molecules of the different individuals. What can be done for that is, you can mix blood from different donors and use it to avoid such problem.
We always transfect into fresh RBCs (certainly less than 2 weeks from blood draw), even if the parasite look like they're growing ok in older blood. For a transfection, you want your parasites growing optimally and they are generally acknowledged to grow best in freshly-drawn blood because, as Praveen notes, blood cells age over time and parasites grow in the youngest cells most efficiently. We also 'feed' them weekly after transfection for the same reason (cells age even faster at 37C than when stored in the fridge). Because blood batches do differ, Kunal's suggestion to mix a couple of different batches may also help your transfections to succeed.
See reference on optimal transfection procedures:
'A quantitative analysis of Plasmodium falciparum transfection using DNA-loaded erythrocytes.'
Hasenkamp, S., Merrick, C.J., Horrocks, P. Molecular and Biochemical Parasitology 187(2), 117-120 (2013).
For general culture growth older blood my be satisfactory but for stable transfection you must use the freshest drawn blood you can get. Depending on the method used for integration, double recombination or piggybac not so much for episomal, they may take a long time for the parasites to become established. New blood is needed to help avoid RBC lysis, because of its age, while you wait for the parasitemia to rise.
Does someone know why is it common use O+ red blood cells for P. falciparum 3D7 culture? Is there some problem in using another blood group?
Dalila - one good reason is that O+ erythrocytes are compatible with all serum types. Parasites will grow in any blood type, but if you chose, e.g., blood group A, you'd need to match the human serum in your culture medium accordingly (which is potentially inconvenient if you use a pooled serum source).
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