F4 80 brilliant violet 421

BSLs purified from perfused brain tissue of moribund C57BL/6 mice on day 6 post-infection were stained with TCRβ-FITC, CD3ε-PE, CD4-PerCP, CD4-PerCP/Cy5.5, CD8-APC/Cy7, CD11b-APC, CD14-PE/Cy7, and F4/80-Brilliant Violet 421™ antibodies purchased from Biolegend (San Diego, CA) and fixable viability dye eFluor^®506 (eBioscience, San Diego, CA). An Amnis^® ImageStream^®X MKII (Amnis Merck-Millipore, Seattle, WA) equipped with five lasers (355, 405, 488, 561, and 642nm) was used for acquisition. A minimum of 30,000 events were collected at 40X magnification using the INSPIRE^® software (Amnis Merck-Millipore, Seattle, WA). Analysis was performed using IDEAS® 6.1 software (Amnis Merck-Millipore, Seattle, WA). Focused events were first gated using the Gradient RMS feature. Singlets were then gated by aggregate discrimination using aspect ratio and area. Analysis was then performed on gated live CD11b^highCD14⁺F480⁺TCRβ⁺CD3ε−CD4−CD8− cells.

Following a FACS buffer soak, single cells from mouse lungs were incubated with antibodies CD11b-PE-Cy7 (101216, BD Biosciences, USA), F4/80-Brilliant Violet 421 (123137, Biolegend, USA), CD86-FITC (105005, Biolegend, USA), Ly6C-APC (128015, Biolegend, USA), and CD206-PE (141705, Biolegend, USA). Stained cells were analyzed by CytoFLEX cytometer (Beckman, USA).

Mice were sacrificed 20 days after tumor cell injection and single cell suspensions were prepared from the tumor-draining lymph nodes (td-LNs), spleens, and one hind limb (for the recovery of BM cells). BM was extracted from the hind limbs as described previously (24 (link)). The cells were re-suspended at 1 × 10⁶ cells/mL and incubated with the following antibodies after non-specific binding was blocked by incubation with Fc receptor block (F16/32): CD11b PE-Cy7, Gr-1 FITC, CD11c APC, F4/80 brilliant violet 421, and CD3 APC-Cy7 (all antibodies were purchased from Biolegend, New Zealand and were titrated on splenocytes and BM cells). Propidium iodide (PI) was added prior to acquiring data using a BD FACSCanto II and FlowJo software (version 9.5.2, TreeStar, Inc., USA) was used to analyze data.