Anti f4 80 alexa fluor 488 and anti cd11b apc cy7

The Kupffer cells and MDMs isolated using magnetic bead separation were washed and resuspended in FACS buffer (PBS, 1% FBS). The cells were then incubated with Fc blocking buffer (BD Biosciences; diluted 1:20) for 10 mins at 4°C. The cells were then rinsed and centrifuged at 300 g for 5 minutes. The cell pellet was then resuspended with anti-F4/80 Alexa Fluor-488 and anti-CD11b APC/Cy7 (BioLegend, San Diego, CA) and incubated for 30 minutes at 4°C. The cells were then washed twice and fixed in formalin (Sigma) for 15 minutes. After cells were fixed, they were washed twice and resuspended in FACs buffer. Fluorescence was then detected using an Attune NxT flow cytometer (Life Technologies, Carlsbad, CA) and the data were analyzed using Attune NxT software.