Monocyte Subsets by Flow Cytometry and Dendritic Cell Flow Cytometry Panel

*Designate clones independently validated by HLDA.
Alexa Fluor^® is registered trademark of Molecular Probes, Inc.

This multicolor flow cytometry panel was validated on human peripheral blood mononuclear cells (PBMCs).

Other Supplies Required

• PBS
• Flow Cytometry Staining Buffer (Catalog # FC001)
• Fc-block (blocking IgG)
• (Optional) Isotype Control Antibodies
• 5 mL Flow cytometry tubes

1. Wash human PBMCs (1 x 10⁶ cells per sample) with 2 mL of Staining Buffer (1X) (Catalog # FC001) or other BSA-containing buffer, by spinning at 300 x g for 5 minutes, using 5 mL flow cytometry tubes. Decant/aspirate supernatant.
2. Fc-block cells with blocking IgG (1 μg IgG/10⁶ cells) for 10 minutes at room temperature.
3. Add previously titrated amount of each of the fluorochrome conjugated antibodies. Vortex tubes.
   
   Monocyte Cell Antibody Concentration Chart  

   Marker	Fluorochrome	Recommended Concentration
   HLA-DR	Alexa Fluor® 405	5 μL/106 cells
   CD86	FITC	10 μL/106 cells
   CD11c	PE	10 μL/106 cells
   CD123	PerCP	10 μL/106 cells
   CD16	APC	10 μL/106 cells
   CD20	Alexa Fluor® 700	5 μL/106 cells
   CD3	Alexa Fluor® 700	5 μL/106 cells
   CD56	Alexa Fluor® 700	0.25-1 μg/106 cells
   CD14	Alexa Fluor® 750	5 μL/106 cells

    
4. (Optional) To a separate tube, add 5 μL of each of the isotype control antibodies. Vortex tubes.
5. Incubate the mixtures for 30-45 minutes at room temperature in the dark.
6. At the end of the incubation, wash with 2 mL of Staining Buffer (1X), by spinning at 300 x g for 5 minutes. Decant/aspirate supernatant.
7. Resuspend the cells in 0.2-0.5 mL Staining Buffer (1X) and acquire on a Flow Cytometer.