Fcγr blocking reagent

Isolation of colonic lamina propria cells was achieved using the Lamina Propria Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s protocol. Leukocyte isolation was performed with 40% / 80% discontinuous Percoll gradient (GE Healthcare). Cells were incubated with FcγR blocking reagent (rat anti-mouse CD16/CD32, BD Biosciences) for 30 minutes on ice prior to incubation with the other antibodies. Antibodies used for flow cytometry analysis are listed in S1 Table. To detect expression of Std fimbriae, flow cytometry was performed as previously described [16 (link)] with modifications. In brief, approximately 5x10⁸ bacteria were fixed with 10% formalin and incubated at room temperature for 20 minutes. After washing with PBS, cells were resuspended in 2% NGS diluted in PBS and incubated at room temperature for 30 minutes. Polyclonal rabbit anti-StdA serum [38 (link)] was added to the cell suspensions following incubation at room temperature for 30 min. After washing with PBS, fluorescently labeled secondary antibodies (Invitrogen) were added. Flow cytometry was performed using a MACSQuant Analyzer 10 (Miltenyi Biotec). The data were analyzed using FlowJo v.10 software (TreeStar).

For FACS analysis, LLC tumors implanted into WT and Rac2-/- mice were excised, minced and digested to single cell population using mixture of enzymes containing 1 mg/ml of collagenase type IV, 10 ug/ml of hyaluronidase type V and 0.01 mg/ml Dnase I for 1 h at 37°C. Cells were solubilized with RBC lysis buffer (Thermo Scientific) and centrifuged. CD11b myeloid cells were purified from tumor cell suspension using the MACS method (Militenyi Biotec) according to the manufacturer's instructions. Briefly, cells were incubated with beads conjugated with anti-mouse CD11b and positively selected on LS columns. The recovered cells were incubated with Fc_γR blocking reagent (BD Bioscience) followed by staining with CD11b (clone M1/70, BD Biosciences) and F4/80 (clone BM8, eBioscience) antibodies. To exclude dead cells, 0.5 µg/ml propidium iodide (PI) was added before data acquisition by FACS caliber instrument (BD Bioscience). CD11b and F4/80 positive cells were sorted by FACS and used for real time PCR studies.