apc- or pe-cy7-anti-cd11b  (Thermo Fisher)

Journal: Nature Communications

Article Title: The ubiquitin ligase Pellino1 targets STAT3 to regulate macrophage-mediated inflammation and tumor development

doi: 10.1038/s41467-025-56440-6

Figure Lengend Snippet: a Representative flow cytometry plots gated on CD45 + living cells isolated from colonic lamina propria of WT and Pellino1-mKO male mice in normal, acute 1.5% DSS, and AOM/DSS groups. Migratory macrophages (CD11b + CX3CR1 int ), resident macrophages (CD11b + CX3CR1 hi ). b (Left) Percentage of CD11b + CX3CR1 hi macrophages, (Right) CD11b + CX3CR1 int macrophage infiltration into the colonic lamina propria in WT and Pellino1-mKO male mice in normal, acute 1.5% DSS, and AOM/DSS groups (H 2 O, n = 4; DSS, n = 5; AOM/DSS, n = 5). These percentages for both CD11b + CX3CR1 hi and CD11b + CX3CR1 int were determined by gating among CD45 + cells. c (Left) Representative images of the wound healing assay. Wound healing assays were performed to examine the migration of BMDMs of WT and Pellino1-mKO under stimulation of 100 ng/mL LPS and 20 ng/mL CCL5 for 24 h. Initial wounded areas were marked with a dashed line. Scale bar = 100 μm. (Right) Quantification of migration areas using ImageJ ( n = 4). d (Left) Representative images of the migration assay. BMDMs from WT and Pellino1-mKO mice were induced to migrate with stimulation from 100 ng/mL LPS, 20 ng/mL CCL5, and 100 ng/mL TGF β for 24 h. Scale bar = 50 μm. (Right) Quantification of migrated cells within the field of view area using ImageJ ( n = 7). e (Left) CFSE-labeled MC38 tumor cells were co-cultured with BMDMs from WT and Pellino1-mKO mice for 6 h in the presence of 100 ng/mL LPS. (Right) Phagocytic index was calculated using the following formula: phagocytic index (%) = (number of F4/80 + CSFE + cells)/(number of F4/80 + cells) × 100 ( n = 5). f Expression levels of mRNA associated with macrophage migration in colon tissues of WT and Pellino1-mKO male mice of normal (WT, n = 5; Pellino-mKO, n = 4), acute 1.5% DSS ( n = 6), and AOM/DSS (WT, n = 5; Pellino-mKO, n = 6) groups were assessed by qRT-PCR and normalized to GAPDH expression. Data were represented as mean ± SD in ( b – f ). All statistical comparisons were made using two-tailed Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: The following fluorochrome-labeled mAbs and staining reagents were used according to the manufacturer’s protocol: PE- or PE-Cy7-anti-B220 (clone RA3-6B2, eBioscience), PerCP-Cy5.5- or PE-Cy7-anti-CD3 (clone 145-2C11, eBioscience), FITC- or PE-Cy7-anti-CD4 (clone RM4-5, eBioscience), PE- or APC-anti-CD8 (clone 53-6.7, eBioscience), APC- or PE-Cy7-anti-CD11b (clone M1/70, eBioscience), PE-Cy7-anti-CD11c (clone N418, eBioscience), PerCP-Cy5.5-anti-CD19 (clone 1D3, eBioscience), PerCP-Cy5.5-anti-CD45 (clone 30-F11, eBioscience), PE-Cy7-anti-CX3CR1 (clone SA011F11, BioLegend), FITC-anti-Ly6G (clone 1AB, eBioscience), APC-anti-Ly6C (clone HK1.4, eBioscience), FITC-anti-MHC2 (clone M5/114.15,2, eBioscience), PerCP-Cy5.5-anti-GR-1 (clone RB6-8C5, eBioscience), PE-anti-F4/80 (clone BM8, eBioscience), FITC-anti-CD80 (clone 16-10A1, eBioscience), PE-Cy7-anti-CD86 (clone GL1, eBioscience), APC-anti-CD206 (clone MR6F3, eBioscience), FITC-anti-CD282 (TLR2) (clone 6C2, eBioscience), PE-anti-CD284 (TLR4) (clone HTA125, eBioscience), PE-Cy7-anti-CD369 (Dectin-1) (clone Bg1fpj, eBioscience), and APC-anti-CD36 (clone HM36, eBioscience).

Techniques: Flow Cytometry, Isolation, Wound Healing Assay, Migration, Labeling, Cell Culture, Expressing, Quantitative RT-PCR, Two Tailed Test