Fitc conjugated cd44 antibody

Tumor tissues were digested with Liberase TM and Dnase I (Roche, Basel, Switzerland) and filtered through 40 μm cell strainers. Cells were initially incubated with CD16/32 antibody to block Fcγ receptors, then stained with 1) the combination of APC-Cy7-conjugated CD45 antibody (BD Biosciences, Franklin Lakes, NJ USA), FITC-conjugated CD4 antibody and APC-conjugated CD8a antibody (BioLegend, San Diego, CA USA), or 2) the combination of FITC-conjugated CD44 antibody, PE-Cy7-conjugated CD62L antibody and APC-conjugated CD4 or CD8a antibody (BioLegend). For Treg discrimination, cells were further fixed and permeabilized (eBioscience, San Diego, CA USA) according to manufacturer’s instructions, stained with PE-conjugated anti-FOXP3 antibody (eBiosciences), and analyzed with FACSAria cytometer (BD Biosciences).

The 10-day-old ICR mice were i.g. inoculated with 10⁵ TCID₅₀ CVA6 strains. At 1 dpi, 3 dpi, 5 dpi, and 7 dpi, mock- and CVA6-infected mice (n = 7–10) were euthanized. The brain and spleen cells were extracted and staining as described previously [31 (link),32 ]. The antibodies used in FACS analysis were purchased from Biolegend Inc., CA, USA: APC-conjugated CD3 antibody (#100312), AF700-conjugated B220 antibody (#103210), BV711-conjugated CD4 antibody (#100549), Biotin anti-CD8 antibody (#100704), FITC-conjugated CD44 antibody (#103006), PE-conjugated CD69 antibody (#104507), FITC-conjugated CD25 antibody (#101907), FITC-conjugated Ly-6c antibody (#128006), PE-conjugated CD62L antibody (#104407), PE-conjugated CD11c antibody (#117308), PerCP/Cy5.5-conjugated Ly-6G antibody (#127615), APC-conjugated F4/80 antibody (#123115), APC/Cy7-conjugated CD45 antibody (#103116), PerCP/Cy5.5-conjugated IgM antibody (#406512), PE/Cyanine7 Streptavidin (#405206), anti-CD16/32 antibody (#101330). LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Thermo Fisher Scientific Co., Ltd, Waltham, USA) was used to distinguish dead cells from living ones. Flow cytometry measurement was performed using the BD LSRFortessa FACS Canto and the data was analyzed by the FlowJoX software.

For immunostaining for flow cytometry, cells were collected using Accutase (Sigma, A6964) and washed once in PBS. 1 × 10⁶ cells per sample were stained with 0.6 µL of APC conjugated CD133 antibody (Invitrogen, 17-1331-81) and 1 µL of FITC conjugated CD44 antibody (BioLegend, 338803) in 100 µL of 1% BSA supplemented PBS solution and incubated in dark for 20 min at room temperature. After incubation, cells were washed once with 5 mL of PBS/1% BSA and analyzed on flow cytometry (BD LSR Fortessa). To gate the CD44^high/CD133⁺ cell population, the median fluorescent intensity (MFI) of CD44-FITC was measured on control replicates (termed parental cells in Fig. S2F, G and overexpression control cells in Fig. S4E, F). The average value of CD44-FITC MFI was used to gate CD44^high cells, and CD133⁺ cells were gated according to the negative staining samples. Gating and cell quantification were performed using BD FACSDIVA, FlowJo.