Ghost dye violet 510

CTL activation was analyzed by measuring the expression of Fas ligand (FasL) perforin and granzyme B, as previously described (41 (link),42 (link)).Fucosylated and non-fucosylated CTLs were co-cultured with T2 cells that were pulsed with E75, CG1, or PR1 (40 ug/mL) at a ratio of 1:1 overnight at 37°C. At the end of the incubation period, the cells were stained with fluorescently-conjugated antibodies targeting CD3, CD8, FasL (BioLegend), CLA, and Ghost Dye Violet 510. After staining for cell surface markers, cells were permeabilized and stained with fluorescently-conjugated antibodies targeting granzyme B and perforin (BioLegend). Staining was analyzed using flow cytometry (BD LSR Fortessa).
To test proliferation, we performed standard CFSE assays. Briefly, fucosylated and non-fucosylated CTLs were labeled with CFDA SE Cell tracer reagent (Invitrogen) following manufacturer’s protocols. Cells were cultured in 96-well round bottom plates at a density of 0.3 × 10⁶ cells in RPMI + 10% FBS in the presence or absence of anti-CD3 and CD28 antibodies (BD Biosciences) to stimulate proliferation. Cells were harvested on days 2, 4 and 5 and stained with anti-CD8, anti-CD4 (Biolegend) and Ghost Dye Violet 510. Live, CD8⁺/CD4⁻ cells were gated on CFSE-positivity, and percent proliferation was calculated by gating on CD8⁺ cells and comparing the CFSE⁺ populations between samples.

Flow cytometric analyses were performed as previously described [57 (link)]. eBioscience antibodies were used unless otherwise specified: TCRβ (H57–597), CD4 (RM4–5), CD8 (53–6.7), CD19 (eBio1D3), CD45 (30-F11), CD11b (M1/70), CD44 (IM7), CD62L (MEL14), CD11c (N418), Ly6G (1A8), PDGFRα (APA5), O4 (O4; Miltenyi), CD13 (R3–242; BD), CD31 (390; BioLegend), MHC1 (28–8-6; BioLegend), and CD16/CD32 (93). Viability was assessed using a Zombie Aqua Fixable Viability kit (cat. no. 423101, BioLegend) or Ghost Dye Violet 510 (cat. no. 13–0870, Tonbo Biosciences).

E2:Kindlin-3 BMT mice were injected with 3×10⁵ B16F10-RFP cells approximately 16 hours prior to harvesting. Lungs were perfused with 10 mL of PBS with 2 mM EDTA. Lungs were diced and enzymatically digested as described in Flow Cytometry. Cells were stained with Live/Dead (Tonbo; Ghost Dye Violet 510), CD31 (Biolegend; 390), and CD45 (Biolegend; 30-F11). Endothelial cells were identified as Live/Dead⁻CD45⁻CD31⁺ and sorted into PBS with 2% FBS. Cells were resuspended in Trizol (Thermo Fisher 15596026) to obtain purified RNA using Zymo Research Direct-zol kit (R2052). cDNA was made using iScript Reverse Transcription Supermix (Bio-Rad 1708840) from equal amounts of RNA input and 20 μL reaction volumes were run on a Roche 480 Lightcycler for 50 cycles. Samples from 3–4 mice per group were run in duplicate. The following Taqman probes from Thermo Fisher were used to quantify gene expression: HPRT (Mm03024075_m1), Il-1β (Mm00434228_m1), IL-6 (Mm00446190_m1), CXCL1 (Mm04207460_m1), IL-15 (Mm00434210_m1), CX3CL1 (Mm00436454_m1), TNF-α (Mm00443258_m1), VEGFR (Mm00438980_m1), ICAM-1 (Mm00516023_m1), ICAM-2 (Mm00494862_m1), VCAM (Mm01320970_m1), M-CSF (Mm00432686_m1), GM-CSF (Mm01290062_m1). Ct values were averaged per sample and gene expression was normalized to HPRT expression using delta delta Ct calculations.