Apc conjugated cd11b antibody

Whole mouse blood was collected and monocytes were separated from erythrocyte and granulocyte on a Ficoll (GE Healthcare) gradient. Separated monocytes were washed with Hank's Balanced Salt Solution and then incubated with 2% goat serum for 10 min, and single stained with allophycocyanin (APC)-conjugated CD11b antibody (1:200, Biolegend, Catalogue #101212) or double stained with CD11b plus PE-conjugated Gr-1antibody (1:600, Biolegend, Catalogue #108408) or PE-conjugated F4/80 antibody (1:100, Biolegend, Catalogue #123110) for 45 min. Cells were then fixed with 1% PFA for 10 min before flow cytometry. Cells population data were obtained on a FACS Calibur cytometer (Becton Dickinson) using the CellQuest software (Becton Dickinson). Data were analysed using FlowJo software (FlowJo, LLC). CX₃CR1^CreER/+:R26^iDTR/+or CX₃CR1^CreER/+:R26^tdTomato/+ mice were used for flow cytometry experiments. Both the EYFP and tdTomato signals were well detectable in the blood monocytes, hence no additional signal amplification strategy was used to detect these signals.

PANC-1, HepG2, SGC-7901, and MDA-MB-231 cells infected with sh-Control or sh-ITGA2s were prepared and stained for flow cytometry with the following antibodies: isotype APC anti-human IgG Fc Antibody (Biolegend, clone HP6017, USA) or APC anti-human CD274 antibody (Biolegend, clone 29E.2A3, USA) or for 30 mins at 4 °C. The cells were then washed with PBS three times, resuspended in 150 μl staining buffer, and analyzed for flow cytometry.
For flow cytometry analysis of the mouse tissue samples, single-cell suspensions were prepared and stained for flow cytometry with the following antibodies: APC conjugated CD45 antibody (Biolegend, 103,112, USA); FITC conjugated CD4 antibody (Biolegend, 100,510, USA); PE-conjugated CD8 antibody (Biolegend, 100,708, USA); APC conjugated CD11b antibody (Biolegend,101,212, USA); and FITC conjugated Gr1 antibody (Biolegend, 108,406, USA). Flow cytometry was performed on BD FACSCelesta (BD Biosciences, USA), and the data were analyzed using FlowJo.