PE Rat Anti-Mouse CD49b

Protocol tips

Upstream tips	Protocol tips	Downstream tips
At the indicated time points, tumor draining lymph nodes(tumor-DLNs) and spleens were harvested from the mice, and minced into small fragments and mechanically dispersed in 3-5 ml cold PBS. After filtering with 70 μm cell strainer (BD Falcon, USA), Single-cell suspensions were adjusted to 1 × 106 cells in 100 μl of PBS.	After this, single-cell suspensions of tumor cells were stained for 30 min on ice with 1 μg of antibodies labeled with fluorochromes and then fixed and permeabilized with a permeabilization buffer (BD Biosciences, USA).	analyzed by FACSCalibur (BD Biosciences, USA). Irrelevant IgG mAbs were used as a negative control. Ten thousands live events were acquired for analysis.

Upstream tips
At the indicated time points, tumor draining lymph nodes(tumor-DLNs) and spleens were harvested from the mice, and minced into small fragments and mechanically dispersed in 3-5 ml cold PBS. After filtering with 70 μm cell strainer (BD Falcon, USA), Single-cell suspensions were adjusted to 1 × 106 cells in 100 μl of PBS.
Protocol tips
After this, single-cell suspensions of tumor cells were stained for 30 min on ice with 1 μg of antibodies labeled with fluorochromes and then fixed and permeabilized with a permeabilization buffer (BD Biosciences, USA).
Downstream tips
analyzed by FACSCalibur (BD Biosciences, USA). Irrelevant IgG mAbs were used as a negative control. Ten thousands live events were acquired for analysis.

Publication protocol

At the indicated time points, tumor draining lymph nodes(tumor-DLNs) and spleens were harvested from the mice, and minced into small fragments and mechanically dispersed in 3-5 ml cold PBS. After filtering with 70 μm cell strainer (BD Falcon, USA), Single-cell suspensions were adjusted to 1 × 106 cells in 100 μl of PBS. After this, single-cell suspensions of tumor cells were stained for 30 min on ice with 1 μg of antibodies labeled with fluorochromes CD45, CD3, CD4, CD8, CD49b, CD25, CD11b, Gr-1 or matched isotypic control antibodies(BD Biosciences, USA) and then fixed and permeabilized with a permeabilization buffer (BD Biosciences, USA). Cells were finally stained with antibody to IFN-γ or FoxP3 for 50 min at 4 °C, washed again, and analyzed by FACSCalibur (BD Biosciences, USA). Irrelevant IgG mAbs were used as a negative control. Ten thousands live events were acquired for analysis.

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