Dear All,
I'm doing the in vitro culture of Plasmodium falciparum (iRBC) with Albumax-I, would like to estimate the phagocytic activity of THP-1 derived macrophages towards iRBC, the literature which I have found were opsonized the iRBC with homologus serum to estimate the phagocytic activity but I am using variable blood groups to grow the culture I couldnt found the homologous serum in this context do the opsonization require or can I directly incubate the pre stained iRBC along with macrophage to estimate the phagocytosis by FACs
Thank you
HI Naidu,
I'm not sure if I fully understand your question. However, my understanding is that macrophages don't typically eat red blood cells well without some opsonin or eat-me signal like externalized phosphatidylserine. I'm not sure about infected red blood cells with Plasmodium, but I imagine that remains the case. Are you using human red blood cells? There are anti-human red blood cell antibodies (Rockland; Abcam) that you might be able to use to opsonize human RBCs. If you can't use IgG-opsonized red blood cells, you may want to try complement-opsonized iRBCs.
Hi Naidu,
If you require opsonization, then your question becomes what?: Does P facipiarum cultured with Abumax affect oposonized red cell phagocytosis?? If that is your question, then you could opsonize using anti-D as most surely, your red cells will be positive for this antigen. These anti-D opsonized cells will be readily phagocytosed by the THP-1 cells but I don't think this is your question.
I think what you are trying to do is similar to what we did:
Wolofsky KT, Ayi K, BRANCH DR, Hult AK, Olsson ML, Liles WC, Cserti-Gazdewich, CM, Kain KC. ABO blood groups influence macrophage-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes. PLoS Pathog. 2012 Oct;8(10):e1002942 Epub 2012 Oct 11
You could use serum from any group O person. If you can ABO type them, that is best but just by chance you'd have about a 40% chance of anyone being group O. I hope this helps.
Hi,
to reliably measure red blood cell phagocytosis by macrophages of any kind,you definitely need antibodies of IgG type.Best is to incubate washed red blood cells with the antiserum for 30 min,then wash again and add antibody-coated red blood cells to macrophages in a ratio of 5 to 10 red blood cells to 1 macrophage.Macrophages might be cultured in medium containing 5% heat-inactivated FCS.Erythrophagocytosis assay can be stopped between 30 to 45 min.To get rid of the non-phagocytosed erythrocytes,you expose the assay culture to hypotonic shock by reducing isotonicity to a third by adding destilled water.After 3-5 min you centrifuge the supernatant away,resuspend the macriphages and then estimate the amount of phagocytosis by measuring the interiorized hemoglobin or other methods as described in our old paper:J.Clin.Invest. 52,812-822,1973.
Diethard Gemsa
We have used PMA-differentiated THP-1 cells (THP-1 macrophages) to measure phagocytosis of trophozoite-stage iRBC in a 96-well plate-based assay (see Clin Vaccine Immunol (2009) 16(3) 312-319). In this assay THP-1 cells are lysed with urea after extensive washing, to release ingested erythrocytes which are quantified by measuring haemoglobin after reaction with 2,7-diaminofluorene. The conditions of the assay were optimised to enable the opsonising activity of malaria immune serum to be quantified, and under these conditions very little phagocytosis occurred in the absence of opsonisation with immune serum i.e. THP1 macrophages do not ingest unopsonised iRBC readily. As a positive control, we used commercially-available rabbit anti-human erythrocyte antibody to opsonise the iRBC, which gives very high levels of phagocytosis. This Ab will opsonise all RBC, so the iRBC would have to be purified first, but this procedure could be used if the aim of the experiments was to investigate THP-1 responses to iRBC.
Hope this helps.
Dear all thanks for your suggestions and sparing valuable time for me
Dear Donald thanks for the article
I have question that from the literature I came to know that plasmodium infected RBC inhibits the phagocytic activity of monocytes my interest of experiment is to enhance the phagaocytic activity by using exogenous chemicals invitro for this I would like to check the amount of P. falciparum phagocytosis by THP-1 derived macrophages in presence and absence of exogenous chemical kindly suggest the feasible experiment to perform phagocytosis and to estimate the amount of phagocytosis.
*when I checked literature they suggested to use fresh autologous serum for phagocytosis (Is it from malaria patient?) I dont have access to patient serum so kindly suggest the alternate and I am using combination of fresh ABO blood groups for parasite invasion
Thank you
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