Streptavidin pecy5

After isolation of the indicated tissues, cells were washed in PBS, and subsequently stained with fixable viability dye eFluor455UV (1:500 dilution) (eBiosciences) and mAb (Clone 93) (ThermoFisher) directed against the FcγRIII/II CD16/CD32 (0.5 µg mAb/10⁶ cells) for 20 min at 4 °C. Cells were washed in PBS/2% FBS supplemented with 0.1% w/v sodium azide and 10 mM EDTA, incubated with the relevant mAb for 20 min at 4 °C and washed again twice. When primary antibodies were biotin-coupled antibodies, cells were incubated with Streptavidin-PE/Cy5 (1:800) (BioLegend) for 20 min at 4 °C. Data were acquired with the BD LSRFortessa^TM X-20 flow cytometer (BD Biosciences) and analyzed using FlowJo software version 10.5.3 (Tree Star Inc.). Cell sorting was carried out with the BD FACSAria™ III equipment. In all experiments, forward scatter (FSC)-H versus FSC-A was used to gate on singlets, with dead cells excluded using the fluorescence-coupled fixable viability dye. Lineage positive (CD3, CD19, NK1.1, Ly6G, and Ter119) cells were removed from further analysis.

H104B5F5/M10 human mammary epithelial cells were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan) and were cultured in α-minimum essential medium containing 10% fetal calf serum (FCS) and antibiotics. MCF-7, MDA-MB-231 and SKBR-3 breast cancer cells were obtained from the same resource and were cultured in Dulbecco’s modified Eagle’s medium/F-12 medium containing 10% FCS and antibiotics. Synthesis of AL10 was described as previous study [15] (link). Sialidase was obtained from Takara Bio Inc. (Shiga, Japan). Human CCL19 and CCL21 recombinant proteins were purchased from R&D Systems (Minneapolis, MN). Anti-ST3Gal-I and anti-CCR7 antibodies were obtained from Epitomics, Inc. (Burlingame, CA). Anti-ERK, p-ERK, p38, p-p38, cyclin D1, cyclin A, and cyclin B antibodies were purchased from Cell Signaling (Danvers, MA). Anti-Caspase 3 antibody was obtained from Genetex (San Antonio, TX). Anti-Bcl-2, Bcl-xL, cyclin E and CDK1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Biotinylated Maackia amurensis lectin II was obtained from Vector Laboratories, Inc. (Burlingame, CA). Streptavidin–PE/Cy5 was purchased from Biolegend (San Diego, CA).