Fitc anti mouse cd206

After the polarisation, THP-1-derived macrophages were harvested and washed in PBS. 1 × 10⁶ cells were then stained using anti-CD11b-PE-Cy7, CD209-FITC, CD163-PE, CD115-PE-Cy7, CD204-FITC, CD206-APC (BD Pharmingen, USA) for 30 min at 4 °C. 1 × 10⁶ cells isolated from fresh xenograft tissues were stained using FITC-anti-mouse F4/80 (Bio-Rad), FITC-anti-mouse CD206 (Biolegend). An isotype-matched IgG was used as negative control. Results were analysed by flow cytometry (FACSCalibur, BD).

0.1 g PVA, MoS₂/PVA, GO/PVA, and MoS₂/GO/PVA hydrogels were firstly added to the 24-well plate, then NE-4C cells were seeded in the wells with a density of 1 × 10⁵ cells. Then 100 ng/mL lipopolysaccharide (LPS) (L4391-1 mg, Sigma-Aldridge) were added to the culture for 24 h, and cells were then collected, washed twice with cold PBS, stained with FITC anti-mouse CD206 (141703 Biolegend, USA) and PE anti-mouse CD11c (101307, Biolegend, USA) in the binding buffer for 30 min, and then detected by flow cytometry (FACS Calibur flow cytometry; BD Biosciences). FlowJo-V10 software was used to analyze the proportion of M1and M2 macrophages in each group.

Tumor was dissected and filtered through a cell strainer (100 µm, Corning, USA) in PBS. Red blood cells (RBC) were lysed using ACK (Ammonium-Chloride-Potassium) RBC Lysing Buffer (0.15 M NH₄Cl, 10.0 mM KHCO₃, 0.1 mM Na₂ EDTA), and the B16-F10 and immune infiltrating cells were kept in PBS. One million cells per well were stained in a round bottom 96 well plate using a two-step staining protocol. First, cells were stained with a live/dead dye (Fixable aqua 405 nm, Invitrogen, USA) at 4°C for 20 min, cells were washed, and 100 μL final volume of a solution containing surface antibodies diluted in staining buffer (1% fetal bovine serum, FBS, 1 mM EDTA, and 0.02% NaN₃ in PBS) were added into each well. After 30 min at 4°C, the samples were washed (2X) and resuspended in staining buffer until acquisition. The following antibodies were used: PerCP-Cy5.5 Anti-Mouse CD80 (Clone 16-10A1 Cat no. 194722), APC-Cy7 anti-Mouse F4/80 (Clone BM8 Cat no. 123118), FITC Anti-Mouse CD206 (Clone C068C2 Cat no.141704), from Biolegend, USA, and PE-Cy7 anti-mouse CD86 (Clone GL1 Cat no. 560582) from BD, USA. Samples were assessed with a FACSCanto II cell analyzer (Becton Dickinson, USA) using DiVA 8 acquisition software and FlowJo 5 V10 (Becton Dickinson, USA) data analysis software.

The brain tissues were obtained from mice after treatment. The brain tissues were cut with scissors, digested with collagenase type IV containing 0.1%, and then prepared into cell suspensions by sieve grinding, and the samples were resuspended by 40% and 70% percoll and centrifuged to collect non-lymphocytes and lymphocytes for use. Lymphocytes were labeled with Percp-cy5.5-anti-mouse CD11b antibody (dilution of 1:50, catalog number: 101228, clone: M1/70, Lot: B353753, Biolegend), FITC-anti-mouse CD206 antibody (dilution of 1:100, catalog number: 141704, clone: C068C2, Lot: B350306, Biolegend) and APC-anti-mouse CD86 antibody (dilution of 1:80, catalog number: 105071, clone: GL-1, Lot: B323580, Biolegend) to detect the polarization of macrophages. The expression level of PDL1 in tumor cells was detected with FITC-anti-mouse CD45 antibody (dilution of 1:100, catalog number:103108, clone: 30-F11, Lot: B363202, Biolegend) and APC-anti-mouse PDL1 antibody (dilution of 1:50, catalog number: 124312, clone: 10F.9G2, Lot: B277024, Biolegend).

Mouse spleen lymphocyte cells were harvested as mentioned previously. Surface biomarkers, PE-anti-mouse CD68, PE/Cy5-anti-mouse CD197, and FITC-anti-mouse CD206 antibodies, were obtained from Biolegend, CA, USA. Nine μL fluorescence conjugated antibodies were mixed with 50 μL cell suspensions and incubated at 4 °C in dark for 30 min. Cells were washed and resuspended in 200 μL analysis buffer (RPMI 1640, 5% FBS, 2 mM l-glutamate, and 2 × Dulbecco’s phosphate buffer saline). Two differentiated cells, M1 (CD68⁺/CD197⁺) and M2 (CD68⁺/CD206⁺) were analyzed using a FACSCalibur™ Flow Cytometer (BD Bioscience, NJ, USA).

For fluorescence-activated cell sorting analysis, 10⁵ peritoneal macrophages or THP-1-induced M0 macrophages (CTR group and UVB group) were isolated and resuspended in 100 µl PBS, and antibodies were added to the corresponding cells. Flow cytometric analysis was performed by FACSCanto II (BD Biosciences) using Flow Jo software. The following antibodies were used: PE/Cyanine7 anti-human CD206 (BioLegend, 321,124), FITC anti-human CD86 (BioLegend, 374,204), APC anti-human CD163 (BioLegend, 333,609), PE/Cyanine7 anti-mouse CD86 (BioLegend, 105,014), FITC anti-mouse CD206 (BioLegend, 141,704), PE/Cyanine7 anti-mouse CD204 (Invitrogen, 2,263,083), and PE anti-mouse CD163 (BioLegend, 156,703).