If isolating mono-nuclear cells and then differentiating them helps, here is a protocol.

Peripheral blood mononuclear cells isolation

Whole blood was obtained by venipuncture of the vena jugularis externa into EDTA (Ethylenediamine tetra-acetic acid) coated vacutainer tubes (Becton Dickinson, Heidelberg, Germany). Peripheral blood mononuclear cells (PBMCs) were isolated from blood by using OptiPrep (60% w/v iodixanol) (STEMCELL technologies, Vancouver, Canada), as per manufacturer’s instructions with minor modifications. A range of density barriers (1.070gm/ml - 1.084 gm/ml) were tested to get maximum yield of monocytes with lowest amount of lymphocyte contamination in the pool of isolated PBMCs. For isolation of monocytes, 1.076 gm/ml density barrier was chosen from the range of 1.070 gm/ml, 1.072 gm/ml, 1.074 gm/ml, 1.076 gm/ml, 1.078 gm/ml, 1.080 gm/ml, 1.082 gm/ml and 1.084 gm/ml density barriers, after optimization. Density barriers (Annexure I) were made as per manufacturer’s instructions, by mixing OptiPrep solution with Complete RPMI1640 medium (Annexure I). Then, 54% (w/v) iodixanol solution was prepared by mixing 5.4 volumes of OptiPrep with 0.6 volumes of 8.5% NaCl solution. One part of 54% (w/v) iodixanol solution was used to adjust the density of whole blood by mixing with 5 parts of whole blood by several gentle inversions. In a 15 ml centrifuge tube, 6 ml of chosen density barrier (1.076 gm/ml) was layered above 4 ml of whole blood and 500 µl of complete RPMI 1640 medium was layered on the top of density barrier. Centrifugation was done carefully without mixing at 700 x g in a swinging bucket rotor for 30 min at 4ºC without brakes during deceleration. Cells floating at the top of density barrier were collected and diluted in 2 parts of complete RPMI1640 medium (RT) immediately. Cells were centrifuged at 1000 rpm at room temperature for 7 min and the obtained pellet was again resuspended in the complete RPMI 1640 medium. The viability of obtained cells was determined by trypan blue exclusion method. Cell suspension was diluted and mixed with trypan blue in 1:1 ratio and 10 µl of suspension was used for counting in Neubauer's chamber. Monocytes were characterized by nuclear stain Hoechst 33342 by observing their ellipsoidal (kidney-bean) shape nucleus. Briefly, 100 µl of cells were mixed with 20 µl of Hoechst 33342 stain for nuclear staining and kept in dark for 15 min. Cells were centrifuged for 2 min at 300 x g and the staining solution was aspirated completely. Further, cells were washed twice with PBS followed by observation of cells at 1000 x magnification under IX-73 Olympus fluorescence microscope.

Note: Solutions should be freshly prepared, mixed thoroughly and maintained at 4ºC before density gradient centrifugation.

Monocyte cell culture and differentiation protocol

Isolated monocytes were seeded at density of 105 cells in 12 well plates, where Poly-D-lysine (PDL)coated 18 mm round coverslips were already placed. For differentiation into macrophages of M2 (anti-inflammatory) phenotype, 500 µl of complete RPMI 1640 was supplemented with 100 ng/ml of M-CSF (Macrophage- colony stimulating factor). Cells were incubated in M-CSF for 24 hrs at 37ºC in 5% CO2, to let adhere monocytes on the PDL coated coverslips in 12 well culture plates. After 24 hrs of incubation non-adhered cells were removed by removing the media from wells and adhered cells were washed twice with 1X PBS to remove any remaining non-adherent cells. Further, cells were incubated for 7 days at 37ºC in 5% CO2 for complete maturation of macrophages. Medium was replaced after every 48 hrs of incubation with washing of cells twice in 1X PBS. Macrophages were assessed for the development of characteristic structure on 1, 3, 5 and 7 days via fluorescence staining of CD163.