70 m filter

Breast cancer 4T1 cells (ATCC, Manassas, VA, USA) were cultured in Minimum Essential Medium (MEM) with 10% FBS, 1% antibiotic/antimycotic, 1% GlutaMAX, 1% NEAA, 1% MEM vitamin, and 1% sodium pyruvate supplements and maintained in humidified atmosphere at 37°C and 5% CO₂. Mice macrophages were obtained by isolation from fresh mice bone marrow. Monocytes were washed twice with PBS and erythrocytes were lysed by red cell lysis buffer (Sigma, USA), and cells were filtered with a 70 µm filter (BD Lifesciences, USA). Differentiation of monocytes to resting macrophages was initiated by 7-day incubation with macrophage medium, containing 10% FBS and 1% penicillin/streptomycin in RPMI 1640 medium.

At the end of the tumor follow-up, the mice were sacrificed and the tumors dissected, cut in small pieces, placed in break-up medium [400U/mL collagen D (Roche) and 5 µg/mL DNAse I (Roche) in Hanks balanced solution (HBSS)] without calcium or magnesium, and incubated for 1 h at 37°C, mixing every 30 min to disaggregate the tumor and the tumor infiltrated cells. The enzyme activity was inhibited by adding stop medium (RPMI 10% FBS supplemented with 5 mM EDTA), and the disaggregated cells were clarified using a 70 µm filter (BD) and washed with RPMI 10% FBS by low centrifugation. The cell pellet was re-suspended in RPMI 10% FBS containing 20 µg/mL DNAse I (Roche) and incubated for 5 min on ice. Subsequently, cells were recovered by low centrifugation, re-suspended in Percoll (Sigma) gradient (40 to 90%), and centrifuged at 1,000 g for 30 min at room temperature. The leukocyte band was obtained from the gradient; the cells were washed once with PBS and finally re-suspended in RPMI 10% FBS medium. They were analyzed for viability using the trypan blue exclusion method in a Neubauer chamber. Afterwards, the cells were treated for flow cytometry analysis.

Spleens and livers were harvested into ice cold FACS buffer (PBS supplemented with 2% FCS v/v) and then emulsified through a 70 µM filter (BD Biosciences), centrifuged and washed in FACS buffer. Liver mononuclear cells were isolated using isotonic percoll gradient. Tissue samples were treated with Red Blood Cell Lysis Buffer (BD Pharmlyse, BD Biosciences). Samples were incubated in CD16/CD32 blocking antibody (clone 93; Biolegend) before staining with appropriate fluorochrome-conjugated antibodies at the listed dilution (Supplementary Table 2). Post-acquisition analyses were performed using FlowJo software V10.0 (Treestar, CA). Cell analysis and sorting were performed using the BD FACS LSR Fortessa^TM or BD FACSAria^TM using FACS Diva Software (version 8.0.1).

Tfh cells, GC B cells, and LLPCs isolated from draining LNs or BMs were analyzed by flow cytometry as previously described (61 (link)). LNs and BMs were collected at 7 or 14 dpi. LNs and BMs were harvested, and single-cell suspensions were obtained by passage of the LNs through a 70-µm filter (BD Biosciences). Red blood cells were removed using lysis buffer (Cat. no. 555899, BD Biosciences Inc., Franklin Lakes, NJ, USA). Cells were washed with PBS and labeled with fluorescence-conjugated antibodies for 30 min at 4°C. After incubation for 30 min at 4°C, the cells were washed twice with PBS containing 0.2% (wt/vol) BSA. Finally, stained cells were analyzed using a BD FACSVerse system. Data were analyzed using FlowJo software (TreeStar, CA, USA).