Fitc conjugated anti mouse human cd11b

Tissue from the spleen tumour was minced and washed repeatedly with PBS. The resulting slurry was subjected to partial enzyme digestion (DNAse, Roche, Cat. no. 10104159001, Basel, Switzerland; Pronase, Roche, Cat. no. 10165921001, Basel, Switzerland; Collagenase, Gibco BRL, Cat. no. 17100, Schwalbach, Germany), passed through a 100 μm nylon mesh, and spun on a density gradient (Ficoll-Paque, Amersham Biosciences, Cat. no. 17544652, Piscataway, NJ). Cells at the interface were collected and washed twice in PBS. The resulting single-cell suspensions were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse/human CD11b (BioLegend, Cat. no. 101206, San Diego, CA), phycoerythrin (PE)-conjugated anti-mouse F4/80 (BioLegend, Cat. no. 123110, San Diego, CA) and Mouse MDSC Flow Cocktail 2 with Isotype Ctrl (BioLegend, Cat. no. 147003, San Diego, CA). Antibodies were diluted 1:100 in flow cytometric buffer (PBS with 5% FBS) and incubated with cells for 1 h at 4 °C. Cells were then washed and resuspended in antibody-free flow cytometric buffer. Flow cytometric measurement of fluorescence emission was performed on a total of 10,000 cells per dissociated tumour tissue by a Beckman/Coulter Epics XL-MCL flow cytometer with EXPO32 software (Beckman, CA).