I am working with bovine monocytes to assay innate immune response against to S. aureus
Hi Marco,
It seems that PMA is not a good option to differentiate bovine monocyte to macrophages. Inasmuch as we did not worked with bovine monocyte/macrophages transition but rather with monocyte-derived macrophages in human THP-1 cell line (please find about it within http://www.xenobiovir.com/), we collected data on other species models, and found the following protocol as regarding bovine mononuclear cells differentiation into macrophages. Bovine mononuclear cells were separated by density gradient centrifugation; whole blood was centrifuged at 300 g for 15 min, Buffy coats were diluted 1:1 in warmed Dulbecco’s phosphate-buffered saline and over-layered onto Ficoll-Paque, then centrifuged at 400 g for 30 min. Contaminating red cells were removed using erythrocyte lysis buffer containing 0.15 M ammonium chloride. Cells were then separated to isolate CD14+ cells. Cells were suspended in RPMI with glutamine supplemented with 10% FCS, containing 200 U/ml penicillin and 200 mg/ml streptomycin at 5–20 x 105cells/ml (1–4 x 105cells/well) in 96-well plates in triplicate for biochemical assays or in 24-well plates for RNA extraction (3–4 × 106 cells/well). Cells were incubated at 37 °C in 5% CO2, medium being replaced every 2 days, until cells achieved 80% confluence and they had matured into monocyte-derived macrophages (MDM), at 6–12 days culture.
MDM or SM were stimulated by addition of 50–2000 ng/ml LPS from E. coli 0111:B4 and/or 20-100 ng/ml bovine IFNγ or with 10-40 ng/ml recombinant bovine IL-4 and/or 10-20 ng/ml recombinant bovine IL-13. Following stimulation for varying periods of time (12–72 h), supernatants (200 μl) were collected and stored at -20 °C for NO, chitinase and IL-6 assays. Cells in 96-well plates were lysed with 100ul 1% Triton X-100 in PBS for 20 min at room temperature and lysates stored at -20 °C prior to arginase assay. Cells in 24-well plates were used for RNA extraction.
Hope the above would help you in your studies.
All the best,
Ilya
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