Splenic CD11b⁺Gr-1⁺ MDSCs from tumor-bearing mice consist of a Ly6G⁺SSC^hi polymorphonuclear and a Ly6G⁻SSC^lo mononuclear fraction. (A) Splenocytes were collected from BW-Sp3 tumor–bearing AKR mice and EG7 tumor–bearing C57BL/6 mice, or their respective naive counterparts and were stained with PE-labeled anti-CD11b and FITC-labeled anti–Gr-1 mAbs. The percentage of CD11b⁺Gr-1⁺ cells is indicated. (Bi) CD11b⁺Gr-1⁺ cells from tumor-bearing mice were purified as described in “Methods.” These cells were stained with PE-conjugated anti-CD11b and either FITC-conjugated anti–Gr-1 or anti-Ly6G. Because the anti–Gr-1 mAb RB6–8C5 recognizes both the Ly6G and Ly6C molecules, costaining with the anti-Ly6G mAb 1A8 was not possible due to overlapping epitopes, as previously reported.²³  Gate R1 represents MDSCs with an intermediate to high Gr-1 expression level, while gate R2 represents the highest Gr-1 expressers. Gate R3 represents Ly6G⁻ MDSC, and gate R4 represents Ly6G⁺ cells. (Bii,iii) Ly6G⁺ and Ly6G⁻ MDSCs were purified as described in “Methods” and were stained with the indicated mAbs. Percentages represent the purity of isolated populations. In addition, a dot plot representing the FSC/SSC profile of the purified cells is shown. Median fluorescence intensity (MFI) of the corresponding Gr-1 expression levels and SSC profiles is given. (C) Purified MDSC fractions were subjected to cytospin and May-Grünwald-Giemsa staining. Pictures from BW-Sp3 and EG7 MDSC subfractions are shown (×40 magnification).