Table 3.

Summary of the monocyte isolation process from buffy coats.

Monocyte isolation step	Procedure	Solutions
1st density gradient: Isolation of PBMC from buffy coats	Pancoll gradient centrifugation (15 min, 1000g, RT, without brake) of a 1:2 dilution of the buffy coat with E/B PBS	-Pancoll (1.077 g/L) -E/B PBS (PBS with 2 mM EDTA and 0.5% BSA)
Washing step (1x)	Immersion of PBMC layer in 50 mL E/B-PBS followed by centrifugation (7 min, 400g, RT, with brake)	-E/B-PBS
Erythrocyte lysis	Resuspension of cell pellet in 2 mL dH2O, 3sec	-Sterile dH2O
Washing step (2x)	Immersion of cell pellet in 50 mL E/B-PBS followed by centrifugation (7 min, 400g, RT, with brake)	-E/B-PBS
2nd density gradient: Separation of lymphocytes	hyperosmotic Percoll gradient centrifugation (15 min, 1000g, RT, no brake) of cell pellet immersed in 12 mL MEM medium, centrifugation of 3 mL cell suspension on 10 mL hyperosmotic solution	-hyperosmotic Percoll solution (19.4 mL Percoll, 16.6 mL dH2O, 4.0 mL 1.6 M NaCl) -MEM medium
Washing step (1x)	Separation of monocytes containing supernatant in 50 mL E/B-PBS followed by centrifugation (7 min, 400g, RT, with brake)	-E/B-PBS
3rd density gradient: isolation of monocytes	iso-osmotic Percoll gradient centrifugation (15 min, 400 g, RT, no brake) of cell pellet immersed in 3 mL MEM medium	-Iso-osmotic Percoll solution (4.15 mL Percoll, 4.85 mL dH2, 1.0 mL 1.5 M NaCl) -MEM medium
Washing step (1x)	Immersion of cell pellet in 50 mL E/B-PBS followed by centrifugation (7 min, 400g, RT)	-E/B-PBS
Preparation of monocyte solution	Immersion of cell pellet in 5 mL MEM medium	-MEM medium
Adjusting the working solution to 1·106 monocytes/mL	Immersion of the calculated amount from the 5 mL MEM cell suspension into the corresponding amount of suppl. MEM medium	-Suppl. MEM medium