Fitc anti mouse cd206 antibody

100 μL of suspension fluid was added to 0.25 μg APC anti-mouse F4/80 antibody (BioLegend), 1.0 μg PE anti-mouse CD86 antibody (BioLegend), and 0.125 μg FITC anti-mouse CD206 antibody (BioLegend), respectively. Homogenized solutions were placed in the dark at 4°C for 30 minutes, followed by addition of 200 μL FACS buffer, and centrifuged at 4°C, 300 ×g for 5 minutes. After removal of supernatant, the cellular solutions were washed with FACS buffer, followed by addition of 1 mL FACS buffer to resuspend cellular samples. Finally, cellular aggregates were broken up and analyzed with BD FACSCanto II, APC anti-mouse F4/80 antibody-specific M1 macrophage (F4/80⁺), APC anti-mouse F4/80 antibody, and FITC anti-mouse CD206 antibody-specific M2 macrophage (F4/80⁺, CD206⁺). FlowJo software was used to analyze the percentage of macrophages in the lung tissues and the proportions of M1 and M2 macrophages in the lung and tumor tissue samples.
Percentage of macrophages: macrophages (F4/80⁺)/cells.
Percentage of M1 macrophages: M1 (F4/80⁺, CD86⁺)/macrophages (F4/80⁺).
Percentage of M2 macrophages: M2 (F4/80⁺, CD206⁺)/macrophages (F4/80⁺).

Tumor tissues were excised for immunofluorescence staining after tumor‐bearing mice were treated with PBS, free DOX, Fe‐MOF and DOX@Fe‐MOF. Briefly, after blocking with 5% bovine serum albumin (BSA) for 0.5 h at 37 °C, the tumor sections were incubated overnight at 4 °C with anti‐CD80 antibody or anti‐CD206 antibody (Abcam, UK) to label the M1‐like and M2‐like macrophages, respectively. After labeling with secondary antibody, the tumor sections were observed under CLSM.
With regard to in vitro polarization, raw 264.7 macrophages were incubated with lipopolysaccharide + murine interferon (IFN)‐gamma (IFN‐γ) for 24 h (M1 polarization), or with interleukin‐4 (IL‐4) for 24 h (M2 polarization) followed by a further 24 h incubation with Fe‐MOF (500 µL, 20 µg mL⁻¹) (M1‐to‐M2 repolarization), and then cellular morphology was observed under the microscope. Raw 264.7 macrophages before polarization treatment were used as controls. For the cytofluorescence assay, treated cells were fixed for 15 min with 4% paraformaldehyde, and then labeled with fluoresceine isothiocyanate (FITC) antimouse CD206 antibody or phycoerythrin (PE) antimouse CD80 antibody (BioLegend), and Alexa Fluor 647 antimouse F4/80 antibody in turn for 30 min, followed by flow cytometry analysis (Gallios).

Cells were harvested by scraping from dishes, and a total of 1 × 10⁶ cells were suspended in 500 μL of 5% FBS/PBS solution, and incubated with APC anti‐mouse CD68 antibody (Biolegend Cat# 137008), PE anti‐mouse CD80 antibody (Biolegend Cat# 104708), FITC anti‐mouse CD206 antibody (Biolegend Cat# 141704) under the manufacturer recommended concentration for 1 h. After being washed with PBS solution 3 times, cells were loaded into the flow cytometer (BD FACSCanto, BD biosciences) and data were analyzed with FlowJo software (v10.8.1). More details are listed in the supplementary key resource Table S1.