I suggest you to use only monocytes , used 24 hours after isolation. It seems to be for me and other researchers a good way to evaluate erythropagocitosis

Thank you very much, but what about erythrocytes during incubation?

I Think that it can be a good idea . Why don't you try and inform how were the results.

There is a lot of variation between donors. Unless you repeat this several times with different donor pairs and see a consistent difference, I would not think that there is a difference between autologous and heterologous erythrocytes. How do you opsonize? Do you use a source of complement? Are natural antibodies in the system? Were the blood groups matched? If so, only AB0 or also others?

Thank you very much, I used immunoglobulins anty RhD antigen to opsonize erythrocytes (DIAMED). I isolated erythrocytes from RhD whole blood

and I have additional question-should I pooled monocytes and erythrocytes?

There are variables that have not been addressed.

The nonopsonized erythrocytes + monocytes were 24 hours post isolation, were they two study groups? one that was nonop rbcs + monos from the same donor,and nonop rbcs + monos from different donors?

The next qroup: nonop rbcs + monos (immediately after isolation) from same donor

and nonop rbcs + monos (immediately after isolation from different donors?

I agree with those ideas above mentioned. I will continue the way suggested.

Dear Anna, I’ll try to answer some of your questions.

First of all, DO NOT pool monocytes or RBCs between different donors, always keep them separately. Monocytes are main mediators of innate immune response: so they are ready to a rapid response to other donors components/cells or pathogens. Moreover, response in different donors is always different. So you can analyse different replicates, from different donors.

Second, I would recommend to use fresh monocytes if the oxidative burst is a key cellular process in your assay (monocytes usually significantly reduce ox burst ability after 12h from isolation ). But if you need the cells to start differentiate towards macrophage-lineage wait after o.night or 24h, and keep them in adhesion with Macrophage-serum free medium (M-SFM from Gibco).

Third, monocytes do not phagocyte RBCs from the same donor; probably except RBCs modified by oxygen radicals or other ageing conditions. Always prepare (through 2-3 washings in cold PBS) RBCs from another donor. I like using IgG antiD, but make sure of course that you have blood from Rh+ donor.

I usually perform erythrophagocytosis with Rh+ RBCs obtained from healthy donors and treated with 0.75 U IgG anti-D/uL packed RBCs for 30 minutes at 37°C (Rhophylac; ZLB Behring). I usually then wash cells once and resuspend in PBS at 30% hematocrit and use within the same day. You can add 10 RBCs per monocytes. Monocytes usually phagocyte 1-3 RBCs. Try different ratio between RBC and monocytes and see how it goes. Leave RBCs for 3h on monocytes, and then wash 3 times (1x PBS + 1x sterile water to lyse remaining non-phagocytosed RBCs in 10 seconds incubation + 1x PBS).

Hope that helps

Valentina

Dear Valentina,

Thank you so much for your important and so helpful information. Could you tell me if you use trypan blue in your assay erythrophagocytosis?

No, I used luminometer assay, that is much more sensitive. Basically, after 3hr phagocytosis, you should wash the cells with RPMI and PBS, then lyse them with a solution of NaOH 0.1N. Then leave the lysed solution at 4 C for 3 hr, then measure the chemiluminescence induced by the ability of iron to oxidize luminol. But I can understand that it might not be easy to have access to this instrument.

1. Opsonized erythrocytes and monocytes  - 11.8% of ingesting erythrocytes

2. Monocytes and opsonized erythrocytes - 26.64% ingesting erythrocytes

3. Nonopsonized erythrocytes (uKKcz) with monocytes - 0.2% intgesting erythrocytes

4. Nonopsonized eryhrocytes (uKKcz) with monocytes -  7.46% ingesting erythrocytes

Interpretations : Opsonized erythrocytes are more prone to get phagocytosed by monocytes.