Contributed by Dr.A.Gratchev

This protocol uses the PBMC fraction enriched in with monocytes by density gradient centrifugations (protocol may be found at www.methods.info). Reduction of the amount of microbeads in comparison to Miltenyi protocol reduces the costs of the experiment.

Note: PBS should be without Ca2+ and Mg2+ !

Protocol

1. Resuspend the cells in buffer (0.5 % BSA, 2 mM EDTA in PBS) at a concentration 10e7 cells per 90 µl.
2. Add 10 µl CD14 micro beads per 10e7 cells.
3. Incubate the suspension for 15 min at a rotator at 4°C.
4. Add 10 ml of the buffer to the cells and centrifuge 10 min at 300 g.
5. Resuspend the cell pellet in 500 µl buffer.
6. Place an LS column in the magnetic separation unit (do not remove the moving part out of the unit!) and wash it with 3 ml of buffer.
7. Change the collecting tube and apply the cell suspension on the column.
8. Collect flow-through.
9. Wash the column 3x with 3 ml buffer collecting flow-through in the same tube.
10. Take the column out of the separation unit and transfer it to a new collection tube.
11. Elute the cells with 5 ml buffer.
12. Count the cells and take an aliquote for FACS analysis.
13. Centrifuge the cells 10 min at 300 g.
14. Resuspend the cell pellet in X-vivo 10 medium at a concentration of 5*10e5 cells/ml.
15. Transfer the cells to the cell culture dishes and add desired cytokines.

Separation units

MidiMACS

QuadroMACS

Images were kindly provided by Miltenyi Biotec

Recommended reading

1. Kzhyshkowska J, Mamidi S, Gratchev A, Kremmer E, Schmuttermaier C, Krusell L et al. Novel stabilin-1 interacting chitinase-like protein (SI-CLP) is up-regulated in alternatively activated macrophages and secreted via lysosomal pathway. Blood 2006; 107(8):3221-3228.
2. Gratchev A, Kzhyshkowska J, Kothe K, Muller-Molinet I, Kannookadan S, Utikal J et al. Mphi1 and Mphi2 can be re-polarized by Th2 or Th1 cytokines, respectively, and respond to exogenous danger signals. Immunobiology 2006; 211(6-8):473-486.
3. Gratchev A, Kzhyshkowska J, Utikal J, Goerdt S. Interleukin-4 and dexamethasone counterregulate extracellular matrix remodelling and phagocytosis in type-2 macrophages. Scand J Immunol 2005; 61(1):10-17.
4. Gratchev A, Kzhyshkowska J, Duperrier K, Utikal J, Velten FW, Goerdt S. The receptor for interleukin-17E is induced by Th2 cytokines in antigen-presenting cells. Scand J Immunol 2004; 60(3):233-237.
5. Kzhyshkowska J, Gratchev A, Martens JH, Pervushina O, Mamidi S, Johansson S et al. Stabilin-1 localizes to endosomes and the trans-Golgi network in human macrophages and nteracts with GGA adaptors. J Leukoc Biol 2004.