Apc conjugated anti mouse cd44

Seven days after tumor re-challenge with B16-OVA tumor cells (2 × 10⁶ cells/mouse), inguinal lymph nodes closest to the tumor were isolated for analysis of effector memory T cell. Strainers (40 μm) were used to dissociate tissues into single cells. Red blood cells were lysed and the resulting cell populations were stained with the following fluorescent antibodies: FITC-conjugated anti-mouse CD3 antibody (BioLegend), PerCP/cyanine 5.5-conjugated anti-mouse CD8a (BioLegend), PE-conjugated anti-mouse CD62L (BioLegend), and APC-conjugated anti-mouse CD44 (BioLegend). The percentage of effector memory T cells [CD3(+)CD8(+)CD44^highCD62^low] was analyzed using a BD FACSCalibur flow cytometer (BD Bioscience), CellQuest Pro and FlowJo software.

BV-510 conjugated anti-mouse CD3 (145-2C11), PerCP-Cy5.5 conjugated anti-mouse CD4 (RM4-5), APC-cy7 conjugated anti-mouse CD8 (53-6.7), FITC conjugated anti-mouse γδTCR (GL3), PE-CD25 conjugated anti-mouse (A7R34), APC-conjugated anti-mouse CD69 (H1.2F3), APC-conjugated anti-mouse CD44 (IM7), PE-cy7 conjugated anti-mouse CD278 (ICOS, 15F9), Percp5.5 conjugated anti-mouse CD16/32 (93), APC conjugated anti-mouse IFN-γ (XMG1.2), PE conjugated anti-mouse IL-4 (11B11), PE conjugated anti-mouse IL-17 (TC11-18H10), PE conjugated anti-mouse IL-10 (JES5-16E3), APC conjugated anti-mouse IL-5(TRFK5), PE conjugated anti-mouse IL-2 (561061), PE conjugated anti-mouse CD154 (MR1), PE conjugated anti-mouse CD183 (CXCR3-173), PE conjugated anti-mouse CD80 (16-10A1), matched control monoclonal antibodies (MG1-45) were purchased from BioLegend (San Diego, CA, USA).

The efficiency of exofucosylation modification on BMSCs was evaluated by the reactivity with mAb HECA452 (which recognizes canonical sialofucosylated E-selectin binding determinants (such as sialylated Lewis X (sLe^x)) both by the flow cytometry and western blotting. In flow cytometry, FTVII-modified and unmodified BMSCs were incubated with APC-conjugated anti-mouse HECA452 (BD Biosciences, New Jersey, USA) and APC-conjugated anti mouse CD44 (BioLegend, Beijing, China) for 30 min at 4 °C. The cells were washed twice with PBS, fixed with 2% paraformaldehyde, and finally tested by flow cytometry. In western blotting, the cell lysate proteins were separated by 10% SDS-PAGE electrophoresis gel and then transferred onto polyscreen polyvinylidene difluoride (PVDF) membranes. For detection of sLe^X, membranes were incubated with mouse HECA452 mAb (NB100-78,039, Novus, Littleton, Colorado, USA), followed by staining with goat anti-rat IgM-HRP (Abcam, Cambridge, UK). The Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level was used as the protein loading control.