Anti cd44 pe

To quantify pancreatic cancer stem-like cells in the bulk Panc-1 cells and lower chamber cells, we measured the expression of the stem cells related molecular marker CD133/CD44 using anti-CD133-PE (Miltenyi Biotech Ltd., Surrey, UK) and anti-CD44-PE (BD Pharmingen, USA). Cells were harvested, disaggregated to a single cell suspension, and stained as described previously [18 (link)].

SPCs (Lipoid S100) were obtained from Lipoid GmbH (Ludwigshafen, Germany) with a purity of 97.6%. It was used as received and stored at −20 °C. GDO was a kind gift from Croda, UK, which contained minimally 95% diglycerides according to the producer. All tool compounds were used as obtained. IR820 was purchased from Sigma. aPD-L1(catalog number BE0101) used in vivo purchased from Bio X Cell. Anti-CD3-PerCP-Cy5.5 (catalog number 551163), anti-CD4-FITC (catalog number 553046), anti-CD8-PE (catalog number 553032), anti-CD4-FITC (catalog number 553046), anti-CD25-APC (catalog number 557192), and anti-Foxp3-PE (catalog number 563101), anti-CD11b-FITC (catalog number 557396), anti-CD11c-FITC (catalog number 557400), anti-CD80-PE (catalog number 560016), anti-CD86-APC (catalog number 553692), anti-LY-6G/LY/6C-PE (catalog number 553128), anti-CD3-FITC (catalog number 553065), anti-CD8-PErCP-Cy5.5 (catalog number 553030), anti-CD62L-APC (catalog number 562910), and anti-CD44-PE (catalog number 559250) were purchased from BD Biosciences.

Mouse gp100_25–33 (mgp100, EGSRNQDWL) and human gp100_25–33 (hgp100, KVPRNQDWL) peptides were synthesized by Peptron (Daejeon, Korea). CD8 microbeads were purchased from Miltenyi Biotec (Auburn, AL, USA). All antibodies used for flow cytometry were purchased from BD Bioscience, including anti-Thy1.1-FITC, anti-CD3-FITC, anti-B220-FITC, anti-CD62L-FITC, anti-Ly-6C-FITC, anti-CD19-PE, anti-CD8-PE, anti-CD44-PE, anti-CD8-PE-Cy5, anti-Thy1.1-PE-Cy5, anti-CD11b-PE-Cy5, anti-CD4-APC, anti-CD8-APC, and anti-CD45-APC. Recombinant human IL-2 (Proleukin) was purchased from Novartis, and the Fc fusion protein of human IL-7 (IL7-Fc), which consist of the extracellular domain of human IL-7 (aa 26–177) fused to the N-terminus of the Fc portion of a mutant human IgG1, was obtained from AdipoGen (Seoul, Korea). The CellTrace CFSE Cell Proliferation Kit was purchased from Invitrogen (Carlsbad, CA, USA).

Cells were trypsinized into single cell suspension and were counted. For staining, samples were usually incubated with antibodies for 30 minutes at 4°C. Unbound antibody was washed off and cells were sorted by flow cytometry no longer than 30 min post staining on a BD Aria III. In order to isolate breast CSCs, the antibodies used were anti-CD44 PE and anti-CD24 FITC (BD Pharmingen). The purity of isolated breast CSCs was determined by standard flow cytometry analysis. The purity of isolated CD44⁺CD24^– CSCs regularly exceeded 98%. As for sorting pancreatic CSCs, cells were stained with APC-labeled CD44 antibodies and PE-labeled CD24 antibodies (BD Pharmingen), as well as BV421-labeled ESA antibodies (Biolegend). The triple positive (CD44⁺CD24⁺ESA⁺) cells and L3.6pl were reanalyzed by flow cytometry analysis using PE-labeled Sox2 antibodies (Biolegend). In sorted cell implantation experiments, CSCs were isolated using APC-labeled antibody against human CD133 (Miltenyi Biotech).

Cell markers were analyzed following a previously published protocol [11 (link)]. Briefly, cells were washed twice in PBS containing 1% bovine serum albumin (Sigma-Aldrich). The cells were then stained with anti-CD13-FITC, anti-CD14-FITC, anti-CD34-FITC, anti-CD44-PE, anti-CD45-FITC, anti-CD73-FITC, anti-CD90-PE, anti-CD105-FITC, anti-CD106-PE, anti-CD166-PE, or anti-HLA-DR-FITC antibodies (all purchased from BD Biosciences, San Jose, CA). Stained cells were analyzed by a FACSCalibur flow cytometer (BD Biosciences). Isotype controls were used in all analyses.