Trustain fcx plus anti mouse cd16 32

All mice used were purchased from Charles River Laboratories. To construct the tumor model, 4T1(1 × 10⁶ cells per mouse) in 25 µL PBS were implanted into the right flanks of BALB/c female mice (6–8 weeks old). Mice were intravenously injected with AMDs (5 × 10⁶ units per mouse) or intratumoral injection of AMDs (1 × 10⁵ units per mouse) on day 7, and each of the treatments was followed by 660 nm (1.0 W cm⁻², 5 min) on day 8. For FACS of t DC cells in TDLNs, TDLNs were collected on day 13. The lymph nodes were dissected from the surrounding fascia, weighed, and minced into pieces by a gentleMACS Dissociator (Miltenyi Biotec). Cell clumps were removed through a 100‐µm cell strainer to obtain single‐cell suspensions. The suspension was centrifuged, and the cell pellets were washed twice with cell staining buffer (Biolegend), blocked 0.25 µg of TruStain FcX PLUS (anti‐mouse CD16/32, BioLegend) Antibody per 10⁶ cells in a 100 µL volume for 5–10 min on 4 °C, and anti‐CD45‐FITC, anti‐ CD11c‐PerCP/Cyanine5.5, anti‐ CD80‐APC, anti‐ CD86‐PE‐Cy7, and anti‐ MHC II‐BV605 antibodies at predetermined optimum concentrations (0.25 µg per 10⁶ cells in a 100 µL volume) were added and incubated on 4 °C for 20 min in the dark. The LIVE/DEAD Fixable Violet Dead Cell Stain Kit (L34963, Invitrogen) was used to determine cell viability during FACS analysis.

B cells from different organs were incubated in TruStain FcX™ PLUS (anti-mouse CD16/32) (BioLegend) blocking reagent for 10 min at 4°C, and then each sample was stained with 1 μg different hashtag antibodies (BioLegend) and incubated for 30 min at 4°C. Cells were washed three times with Cell Stain Buffer (BioLegend) and resuspended with PBS + 0.04% BSA (Sigma). According to the cell number count, B cells from different organs were mixed at the ratio of 1:1 and cell concentrations were adjusted to about 1,000 cell/µl. For 10× Genomics scRNA-seq, cells were loaded onto a 10× chromium controller to generate Gel beads in emulsion using the 10× genomics Single Cell 5′ Library & Gel Bead Kit. Gene expression libraries were constructed according to instructions from 10× genomics. Three libraries were generated that measure (1) mRNA transcript expression (RNA), (2) mouse-specific hashtag oligos (HTO), and (3) BCR library. Libraries were sequenced by the NovaSeq 6000 sequencing system (Illumina, San Diego, CA, USA).

Animals were transcardially perfused with precooled PBS. Then, the whole brain was removed and placed in precooled complete DMEM to further isolate the hippocampus under a stereomicroscope. The hippocampi were scissor-minced and manually homogenized. The tissue was subsequently dissociated by adding accutase (Sigma-Aldrich) and incubated at 37 °C, 5% CO₂ in a cell culture incubator for 15 min. The reaction was stopped by adding serum-containing culture medium. Single-cell suspensions were filtered with a 70-µm nylon mesh. Density gradient centrifugation using Percoll (Biosharp) was performed to isolate the mononucleated immune cells. Samples were washed with PBS three times. Fc receptors were blocked by TruStain FcX™ PLUS (anti-mouse CD16/32, Biolegend) for 5 min on ice. PE anti-mouse CD3ε antibody (Biolegend), FITC anti-mouse CD4 antibody (Biolegend) and APC anti-mouse CD8a antibody (Biolegend) were added for 20 min at 4 °C and protected from light. Isotype-matched antibodies were used as controls. After cleaning the cells with PBS three times, samples were run on a BD LSRFortessa cell analyzer.