I need to know an easy protocol of Hetrophiles and Macrophages extraction from blood specially from Avian/chicken. and also want to know some related knowledge which can help me to test macrophages and neutrophiles against pathogenic APEC strains.
Hello Sayed,
Here is the method we used in cattle and pig, should work in avian too. (You can get the monocytes from blood but not the macrophase, monocytes convert to macrophase in tissue/you can culture monocyte to get macrophase).
1. Collect the blood with anticoagulant (i feel heparin is better than EDTA).
2. Centrifuge the blood at 200-500g for 15 min.
3. Collect buffy layer for monocytes, and redblood cells portion for heterophils.
Heterophils: Lyse the RBS using comercial RBC lysising solution (we made our own). After lysis you will get heterophils (higher proportion).
Monocytes: Dissolve your monocytes in PBS/PBS-heprin. and layer over 65% percoll and centrifuge for 30 min. You will get write layer of PBMCs. If you culture your PBMCs in RPMI media, Monocytes will attach the plastic surface while lymphocytes flots.
Hope it will work for you
Thanks
can any one send me the reference paper about heterophil count in poultry.
Sayed Abdullah
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