Fitc anti human cd68

MDMs were cultured in 12-well plates and exposed to an M1 polarization stimulus using E. coli lipopolysaccharide (LPS) (Sigma, 20 ng/ml) plus recombinant human interferon (rh-IFN)-γ (Fisher, 20 ng/ml) and into M2a using rhIL-4 (20 ng/ml) for 48 h per prior methods (2 (link), 27 (link)). Resident cells without any stimulation were used for baseline comparison. Aliquots incubated in human Fc-γ Block (Biolegend, 422301) for 30 min on ice and then stained using phycoerythrin (PE) anti-human CD80 (Biolegend, 305208, Clone 2D10) and phycoerythrin cyanine tandem conjugate (PE/Cy7) anti-human CD11b (Biolegend, 101216, Clone M1/70) or allophycocyanine (APC) anti-human CD163 (Biolegend, 326510, Clone RM3/1) and allophycocyanin cyanine 7 tandem conjugate (APC/Cy7) anti-human CD206 (Biolegend, 321120, Clone 15-2). Next, FITC anti-human CD68 (Biolegend, 333806, Clone Y1/82A) intracellular staining was performed post-fixation and permeabilization according to the manufacture's recommendations. Stained cells were gated via viable CD11b⁺subsets and identified by CD68CD80 (M1) and CD163CD206 (M2) markers using FlowJo (Tree Star).

NPC cells were co-cultured with macrophages for 48 h. Then the macrophages were trypsinized and resuspended in phosphate buffer saline to a concentration of 1 × 10⁷/ml, and dispensed into 100 μl into the flow cytometry tubes. Overall, 5 μl extracellular antibody APC antihuman CD206 (321109, Biolegend, CA, USA) was added and kept still for at least 30 min, then 5 μl intracellular antibody FITC antihuman CD68 (333805, Biolegend, CA, USA), and PE antihuman CD86 (374205, Biolegend, CA, USA) were incubated in the dark for at least 30 min after permeabilization. Then protein expression was detected by flow cytometry (Beckman Coulter, Brea, CA, USA).
The xenografts were cut and dispersed into single cells, filtered through a 300-mesh filter cloth and centrifuged. The cells were resuspended in PBS to a concentration of 1 × 10⁷/ml, and dispensed into 100 μl into the tubes. The following steps were the same as the former cell experiments.

U937 cells were induced to M2 phenotype by treatment with phorbol 12-myristate 13-acetate (PMA; 100 nM; Sigma) for 24 h followed by interleukin-4 (IL-4; 20 ng/mL; Cell Guidance System) in combination with interleukin-10 (IL-10; 20 ng/mL; Cell Guidance System) for 48 h (called M2 Mφs^Cyto). Alternatively, M2 macrophages were generated by treatment with 10% of HepG2/U937 coculture CM for 48 h (called TAMs^CM). The cells were stained with FITC anti-human CD68 (BioLegend, #333806) and PE anti-human CD204 (BioLegend, #371904) in 4 °C for 30 min. The M2 phenotype was determined by detecting CD68/CD204 expression using a FACS Calibur flow cytometer (BD Biosciences).