Lsrfortessa h0081

For detection of peripheral blood leukocytes, blood was collected by retro-orbital puncture and red blood cells were lysed with ACK. For detection of cells in spleens, tissue was processed into single-cell suspension using a gentleMACS dissociator before ACK lysis. Neutrophils and monocytes were identified using the following gating scheme: Cells were first gated based on forward scatter-A and side scatter-A, singlets were gated based on forward scatter-W and side scatter-A, and then neutrophils (Ly6G⁺CD11b⁺), non-neutrophils (Ly6G⁻), and monocytes (CD11b⁺CD115⁺) identified. For some samples, cells were also stained with mAb specific for myeloperoxidase or CCR2. B cells, NK cells, CD4 T cells, CD8 T cells, and Ag-experienced CD4 and CD8 T cells were identified using the following gating scheme: Cells were first gated based on forward scatter-A and side scatter-A, singlets were gated based on forward scatter-W and side scatter-A, and then live cells (ghost dye⁻) were gated. B cells (CD3⁻CD19⁺), NK cells (CD3⁻NK1.1⁺) and T cells (CD3⁺NK1.1⁻) were identified. Among the gated CD4⁺ and CD8⁺ T cells, Ag-experienced CD4 T cells (CD11a^hiCD49d^hi) and Ag-experienced CD8 T cells (CD8a^loCD11a^hi) were determined. All samples were acquired on Fortessa X20 or LSRFortessa H0081 flow cytometers (BD) and analyzed using FlowJo software.

Rhodamine-labeled NPs were used for this study. LM2 cells suspended in FACS buffer (200,000/150 μl) were incubated with 50 μg suspension of NPs on a rotating platform at 37°C for 1 h (n = 2). At the end of 1 h, the cells were washed twice using FACS buffer, fixed using 4% v/v formaldehyde in FACS buffer, washed once, and then analyzed using flow cytometry (BD LSRFortessa H0081). In order to normalize data to rhodamine loading, fluorescence of the initial NP suspensions was measured using the IVIS Spectrum in vivo Imaging System (Caliper Lifesciences, MA, USA) or Spectramax i3x (Molecular Devices, CA, USA) fluorescence plate reader.