Quantification of Marginal Zone Macrophages

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.