Anti cd86 fitc

BTK inhibition with PF-06250112 was evaluated on splenocytes labeled with anti-CD86-FITC (BD Pharmingen, San Jose, CA, USA), and anti-B220-PE (BioLegend, San Diego, CA, USA). Phenotypic analyses were performed using anti-CD4-Alexa 700, anti-CD19-Pacific blue (Biolegend), anti-CD45-APC (eBiosciences, San Diego, CA, USA); anti-CD3e-FITC, anti-CD11b-PE (BD Pharmingen), anti-CD21-APC Cy7, anti-CD23-PE Cy7 and anti-GL7 Alexa 488 (Biolegend). Fluorescence activated cell sorting analysis was done on live cells using BD LSRII and FACSDiva software.

For confocal imaging, macrophages were plated in coverslip-bottom culture chambers (LabTEK) to confluency and fixed in 1x PBS with 4% w/v paraformaldehyde for 30 min at room temperature. The cells were then washed with PBS three times and incubated with anti-CD14-FITC, anti-CD86-FITC, anti-F4/80-PE, or anti-CD301-APC (BD Pharmigen^TM, San Diege, CA, USA) for 1 h at room temperature, respectively. Cells were then washed with PBS three times and mounted with antifade mounting medium with DAPI (Vectashield, Burlingame, CA, USA). Confocal microscopy was then performed with an Olympus Fluoview FV10i automated confocal laser-scanning microscope (Olympus, Tokyo, Japan).

Single-cell suspensions were generated from spleen and liver of tumor-bearing mice. The following anti-mouse antibodies were used: anti-CD45-BUV395, anti-CD3E-PE/CF594, anti-CD44-PE, anti-CD4-APC/H7, anti-CD8-APC, anti-CD69-FITC, anti-CD86-FITC, anti-CD11c-PE/Cy7, anti-Gr1-PE, anti-CD11b-APC, anti-CD19-PE/Cy7, anti-NK1.1-PerCP/Cy5.5 (BD Bioscience, San Diego, CA), anti-Ly6C-BV421, anti-Ly6G-AF700, anti-CD4-FITC, anti-CD62L-PerCP/Cy5.5, anti-NKG2D-FITC, anti-F4/80-PerCP/Cy5.5, anti-IFN-γ-PE and anti-TNF-α-PE/Cy7 (Biolegend, San Diego, CA). Anti-mouse CD16/32 FcR block antibody was purchased from Biolegend (San Diego, CA). For intracellular cytokine staining, cells were stimulated with Cell Activation Cocktail (Biolegend, San Diego, CA) for 4 hours and stained with surface antibodies. Then cells were fixed, permeabilized and stained with intracellular cytokine antibodies. Cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA).^{29 (link)} Data were analyzed using FlowJo software (Tree Star, Ashland, OR).

Immature BMDCs were generated according to a standard protocol (BMDC isolation protocol, Abcam, USA). Bone marrow cells were isolated from the femurs and tibias of C57BL/6 mice (male, 8–10 weeks old) on day 0. After filtration on a 40 μm cell strainer and red blood cell lysis, bone marrow cells were then seeded in 100-mm Ultra-Low Attachment Culture Dish containing 10 mL RPMI-1640 medium supplemented with 10% FBS, 20 ng/mL GM-CSF and 10 ng/mL IL-4. The cells were cultured at 37°C in an incubator containing 5% CO₂. On day 3, an additional 10 mL of RPMI-1640 medium was added, and then half of the culture medium was changed on day 6. During cultivation, the same concentrations of GM-CSF and IL-4 were maintained as above.
On day 8, both the semi-suspended cells and loosely attached cells were gently collected and plated into 6-well plates. The cells were treated for 48h with (1) PBS, (2) PLGA, (3) PLGA-ICG, (4) PLGA-R848, (5) PLGA-ICG-R848, (6) R848 (5 μg/mL) and (7) LPS (0.1 μg/mL). LPS at 100ng/mL was used as the positive control.
On day 10, BMDCs were stained with anti-CD11c PE (BD Pharmingen™, #557401), anti-CD86 FITC (BD Pharmingen™, #553691) and anti-CD80 APC (BD Pharmingen™, #560016), and then analysed by flow cytometry (Miltenyi Biotec, Germany).