Anti cd11b alexafluor 488

Cells were harvested, counted and 1 × 10⁶ cells were resuspended in 100 μL PBS containing 1% Bovine Serum Albumin (BSA). Pre-conjugated antibodies were added at their optimal concentrations and incubated on ice for 20 min. Cells were subsequently washed in 2 mL PBS (containing 1% BSA) and centrifuged at 400×g for 5 min. Cell pellets were resuspended in 200 μL PBS with 1% BSA. In most experiments cells were stained with a live/dead cell viability stain such as DAPI or 7-AAD. The following anti-mouse antibodies were used: anti-F4/80 APC (BioLegend), anti-CD11b Alexafluor 488 (BioLegend). The following anti-human antibodies were used: anti-CD93 PE (eBioscience), anti-25F9 APC (eBioscience), anti-CD11b Alexafluor 488 (BioLegend).

Tumor samples were collected individually, and cell suspension was obtained after mechanical dissociation and incubation with collagenase D (Roche, Basiléia, Swiss) at a concentration of 0.22 U/mL per sample at 37 ºC for 40 min. Then, samples were washed twice with PBS 1X (pH 7.4), filtered in a 70 µm cell strainer (BD Biosciences), and resuspended in solutions containing anti-CD45-PerCpCy5.5 (BioLegend), anti-CD11c-PE (BD Biosciences), MHC-II-BV421 (BioLegend), F4/80-FITC (Thermo Fischer Scientific), anti-CD86-APC (BioLegend), anti-CD11b-Alexa-Fluor488 (BioLegend), anti-Gr-1-PE (BD Biosciences), CD8a-BV605 (BioLegend), and anti-IFN-γ-PE (BioLegend) mAbs for 30 min at 4ºC. For analysis of E7-specific intratumoral CD8 T cells, samples were stained with the APC-labeled H-2Db E7-specific dextramer (Immudex, Copenhage, Denmark), and subsequently stained with anti-CD8a-Pacific Blue (BioLegend). After two washes, cells were resuspended in PBS, acquired in a LSR Fortessa flow cytometer (BD Biosciences), and analyzed using the Flow Jo software (BD Biosciences).