Percp cy5.5 anti mouse cd11c

BMDCs prepared as described earlier were harvested and stained with PerCP/Cy5.5 anti-mouse CD11c, PE anti-mouse CD80, APC anti-mouse I-A/I-E (MHC II), FITC anti-mouse F4/80, or the corresponding isotype control (BioLegend) for 25 min at 4 °C. After washing, cells were analyzed with flow cytometry. Data analysis was performed by using FlowJo software (Tree Star, Ashland, OR, USA), and the results were reported as mean fluorescence intensity.

On day 45, spleen single-cell suspensions were collected from the five groups of mice, filtered with a cell strainer, and stained with PerCP/Cy5.5 anti-mouse CD11c, PE anti-mouse CD80, APC anti-mouse I-A/I-E (major histocompatibility complex II, or MHC II), or the corresponding isotype control (BioLegend, San Diego, CA, USA) for 25 min at 4 °C. After washing with wash buffer, cells were analyzed by using flow cytometry.

Mice were killed and the small intestine was removed by cutting below the stomach and above the caecum. Intestinal contents were cleared by flushing with PBS. Intestines were cut into small pieces and put into 40 ml digestion buffer containing 5 mM EDTA, 1 mM DTT, 0.2 g dispase, and then shake for 40 min at 200 rpm, 37 °C. After incubation, filter the cell solution through a 40 μm cell strainer and pellet the cells by centrifugation. The cells were further applied with percoll gradient separation to isolate the leukocytes. Cells were stained with DAPI (Thermo Scientific), BUV395-anti-mouse CD45 (Clone: 30-F11, BD Bioscience), PerCP/Cy5.5-anti-mouse CD11c (clone: N418, Biolegend), PE-Cy7-anti-mouse CD11b (clone: M1/70, Biolegend) for 30 min before sorted on the BD FACSAria™ Fusion Flow Cytometers (BD Biosciences).
Feces from control or tumor-bearing mice were suspended in sterile PBS (100 mg/ml), homogenized, and filtered through 40 mm cell strainer to remove aggregates and used to stimulate the LP phagocytes at a 1:200 dilution for 4 h. After that, the medium was replaced with fresh medium containing 100 mg/ml gentamicin and continued culture for 16 h. The supernatant was collected for IL-1β measurement.

Alkaline Phosphatase (AP) anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG2c and IgG3 were acquired from Southern Biotech, while AP anti-mouse IgG was purchased from Bio-rad. BV421 anti-mouse I-A^b, PE anti-mouse I-A^d, BV421 anti-mouse H-2K^b, BV421 anti-mouse GL7, BV480 anti-mouse IgM, BV510 anti-mouse CD38, BB515 anti-mouse CD19, BV650 anti-mouse RORγt, PE CF594 anti-mouse GATA3, BB515 anti-mouse CD8α, and Alexa Fluor 647 (AF647) anti-mouse Bcl6 were obtained from BD Biosciences. FITC anti-mouse I-A^k, AF700 anti-mouse CD45, BV510 anti-mouse H-2K^d, PE anti-mouse H-2K^k, BV605 anti-mouse CD19, AF700 anti-mouse T cell receptor (TCR), AF700 anti-mouse CD11b, AF700 anti-mouse CD11c, BV650 anti-mouse IgD, BV421 anti-mouse CD3, BV510 anti-mouse CD4, BV605 anti-mouse Tbet, BV785 anti-mouse CD25, PerCP Cy5.5 anti-mouse CD19, PerCP Cy5.5 anti-mouse CD11c, PerCP Cy5.5 anti-mouse CD11b, and AF700 anti-mouse CD44 were purchased from Biolegend.