Abundance of immune cell subsets in CIT6-YFP and CIT9-RFP subcutaneous tumors and establishment of mixed tumors. CIT6-YFP and CIT9-RFP tumors were injected into homozygous Confetti mice and harvested at 10 mm for immune cell analysis. (A) Representative image of CD45 stain and quantification of CD45⁺ total immune cells per field in CIT6-YFP and CIT9-RFP tumors as measured by immunofluorescence staining. CD45⁺ cells were counted in 31 (CIT6-YFP) and 23 tiles (CIT9-RFP) of a tile scan image. The data are a representation of eight CIT6 tumors and six CIT9 tumors analyzed. Scale bars are 200 µm. (B and C) (B) Abundance of B cells and NKp46⁺ lymphocytes and (C) myeloid cell types as measured by flow cytometry. n = 9. The data in B and C presented as count per mg and % CD45⁺ cells are a representation of one and three independent experiments, respectively. (D and E) (D) Frequency of mixed-population tumors and (E) representative fluorescent stereomicroscope whole-tumor images of mixed-population tumors formed in Confetti, FVB/N, and NOD/SCID/IL-2Rγ null mice (n = 12, 8, and 8, respectively). Scale bars in E are 1 mm. The data are a representation of one (FVB/N and NOD/SCID/IL-2Rγ null mice) and three (Confetti) independent experiments, respectively. (F) Flow cytometric gating strategy of main immune cell types. Immune analysis of a subcutaneous CIT6-YFP tumor is shown as representative data. Statistical significance was determined by Student’s t test in all data. *P < 0.05, **P < 0.01, and ***P < 0.001.