Macrophages in the wounds were isolated by enzymatic digestion [29 (link)]. Cell surface antigens were blocked with Fc block (20 ug/mL; BD Biosciences), incubated with fluorophore-conjugated antibodies or isotype control antibodies for 1h. Fluorophore-conjugated primary antibodies were from BioLegend: F4/80-FITC (#123122), CD11c-phycoerythrin (#117308) and CD206-Alexa Fluor 647 (#141712). Cells were washed and centrifuged at 500 g for 5 minutes and re-suspended in 1 ml washing buffer for FACS Calibur analyses using WinMDI software and gating strategy was described as previous [28 (link)]. Briefly, wound macrophages were collected and mononuclear cells were gated for FSC/SSC. Dead cell fractions were removed. The gated CD11b+F4/80+ cells were examined with anti-CD11c (as M1) or anti-CD206 (as M2) antibody.
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Isolation and Characterization of Wound Macrophages
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Alexa fluor 647
Antibodies
Antibodies isotype
Antibody
Buffer
Cd11b
Cell surface antigens
Cells
Digestion
Enzymatic
Fitc
Macrophages
Phycoerythrin
Wound
Corresponding Organization :
Other organizations : University of Minnesota, Twin Cities Orthopedics
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Variable analysis
independent variables
- Enzymatic digestion to isolate macrophages from wounds
- Expression of cell surface markers F4/80, CD11c, and CD206 on wound macrophages
- Fc block (20 ug/mL; BD Biosciences) to block cell surface antigens
- Isotype control antibodies used for comparison with fluorophore-conjugated primary antibodies
- Positive control: F4/80-FITC, CD11c-phycoerythrin, and CD206-Alexa Fluor 647 antibodies to label specific cell surface markers
- Negative control: Isotype control antibodies to account for non-specific binding
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