Lsr x 20

To measure the cell surface expression of CD47, HER2, EGFR, and CD20, cells were stained with conjugated primary antibodies (BD Biosciences) for 30 minutes at 4°C, washed, and resuspended in staining buffer. Macrophages were incubated with anti-CD16/32 (BD Biosciences) for 15 minutes at room temperature to block nonspecific binding and stained with CD14, CD68, CD80, CD163, CD206, PD-L1, and HLA-DR (BD Biosciences). Cells were acquired using a BD LSR X-20, and the data were analyzed using FlowJo software.

Two-hundred-microliters of CC81-GREMG cells (1 × 10⁶ cells) or 10-fold concentrated BLV-containing supernatant were supplemented with 20 µg/mL of BSI-625, -679, or DMSO, and allowed to stand for 20 min at 25 °C. BSI-625- or -679-treated cells were mixed with DMSO-treated supernatant, and DMSO-treated cells were mixed with BSI-625- or -679-treated supernatant. DMSO-treated cells mixed with DMSO-treated supernatant were used as the 100% control, and cells mixed with the medium were used as the 0% control. The mixture was incubated at 4 °C for 2 h, and cells were washed five times with cold PBS. Thereafter, cell-surface bound virus was stained with anti-BLV monoclonal antibody (mAb) (BLV-1: VRMD, Pullman, WA, USA) and Alexa-647 conjugated anti-mouse mAb (Invitrogen). Cell-surface fluorescence was measured by BD LSR X-20 (BD Bioscience, San Jose, CA, USA) and the mean fluorescence intensity of cells was compared with that of DMSO-treated controls.

Single-cell suspensions from CNS tissues were stained for viability with aqua, washed, and subsequently surface-stained with a panel of fluorescently conjugated antibodies against CD45.1 (A20), CD3 (17A2), CD4 (RM4-5), CD19 (1D3), B220 (RA3-6B2), Gr-1 (RB6-8C5) or Ly6G (1A8), Ly6C (HK1.4), CD11b (M1/70), and CD11c (N418). Cell suspensions from T cell stimulation were surfaced-stained for CD4 (RM4-5), permeabilized with CytoFix/CytoPerm (BD Biosciences) for 20 minutes at 4°C, and subsequently stained intracellularly with GM-CSF (eBioscience, Thermo Fisher Scientific), IFN-γ (eBioscience, Thermo Fisher Scientific), and IL-17A (eBioscience, Thermo Fisher Scientific) (Supplemental Table 1). Cells were acquired on a BD LSR X20 using FACSDiva software.