Cd11b fitc clone m1 70

Single cell suspensions were prepared from freshly isolated HER2-LLC1 tumors and spleens at sacrifice. Tumors were minced in small pieces and digested with collagenase (1 mg/ml) for 1.5 h at 37°C. The resulting cell suspensions were passed through 70 μm cell strainer and rinsed with flow cytometry buffer. Spleens were processed as described above, and then treated as the tumor samples. Red blood cells in spleen and tumor specimens were lysed by means of ACK buffer, samples were pelleted and resuspended in flow cytometry buffer. Subsequently for each sample 2x10⁶ cells were blocked with α-CD16/32 Ab (clone 93, eBioscience), and then reacted with the antibodies CD4-FITC (clone GK1.5, eBioscience), CD8a-PE (clone 53–6.7, eBioscience), CD45-FITC (clone 30-F11, eBioscience), CD45-Percp-Cy7 (clone 30-F11, eBioscience), CD335-PE (clone 29A1.4, eBioscience), FoxP3-PE (clone 150d/e4, eBioscience), CD11b-FITC (clone M1/70, eBioscience), PD-L1-APC (clone MIH5, BD), CD141-PE (clone LS17-9, eBioscience) and CD69-PercP (clone H1-2F3, eBioscience). Data were acquired on BD C6 Accuri. Only samples which provided at least 100000 events were included in subsequent analysis.

Flow cytometry assays were performed as previously described [18 (link)]. Briefly, cells were blocked with Fc blocking antibody (Clone 2.4G2, Cat Nu 553142, BD Biosciences, San Jose, CA) and stained with monoclonal antibodies recognizing ferret CD4 (clone 02 –PE, Sino Biological Inc., Beijing, China), or cross-reacting with ferret CD8 (clone OKT8 –eFluor 450, eBioscience, San Diego, CA), CD11b (clone M1/70 –FITC, eBioscience), anti-MHC class II (clone CAT82A –unconjugated, Kingfisher Biotech, St. Paul, MN), CD3 (clone PC3/188A –FITC and AlexaFluor 647, Santa Cruz Biotechnology, Santa Cruz, CA), and CD79a (clone HM47 –PerCP-Cy5.5, eBioscience). The anti-MHC class II antibody was biotinylated (cat# 130-093-385, Miltenyi Biotec, San Diego, CA) prior to use and detected with streptavidin-eFluor 450 (eBioscience). Unstained cells and isotype controls (eBioscience, San Diego, CA) were included for all antibodies. Data were acquired using a FACSCanto II flow cytometer (BD Bioscience, San Jose, CA), and analyzed using FlowJo software (Tree Star, Ashland, OR).

The method of Rose et al. was used to quantify numbers of marginal zone macrophages [24 (link)]. Briefly, spleens were harvested and a cell suspension was made after dicing spleens and treatment with DNase I and collagenase D. 1 × 10⁶ to 2 × 10⁶ cells were pelleted at 1500 rpm for 3 min and supernatant discarded. Cell suspension was incubated with 0.5 µg Fc Block (BD Biosciences, San Jose, USA) for 10 min at RT, followed by a wash with FACS buffer (0.5% BSA in saline). Supernatant was discarded once again and the following antibodies were added to each well: CD45R (B220) clone RA3-6B2 PE-Texas Red (1:400, BD Biosciences), Ly6C clone AL-21 APC-Cy7 (1:500, BD Biosciences), Ly6G clone 1A8 PE (1:400, BD Biosciences), NK1.1 clone PK136 APC (1:300, BD Biosciences), CD11b clone M1/70 FITC (1:160, eBioscience, San Diego, CA, USA), CD11c clone HL3 PE-Cy7 (1:125, BD Biosciences) with 20% 7-AAD in FACS buffer. The stain and cell mixture was incubated for 20 min on ice in the dark, and washed twice in FACS buffer. Lastly, samples were resuspended in FACS buffer and kept on ice, in the dark, until being run on a FACS Aria flow cytometer (BD Biosciences). Mean and standard deviation were calculated for PBS and the 33 μL treatment group, and Student’s t-test was used to determine if differences between these groups was significant.