Pe conjugated anti cd45 5b 1 antibody

For CTC isolation, CELLection™ Pan Mouse IgG Dynabeads^® (4.5 μm; Thermo Fisher Scientific Inc., Taipei City, Taiwan) in combination with an anti-EpCAM antibody [Ber-EP4] (ab7504; Abcam, Cambridge, UK) were used. Blood samples were lysed with red blood cell (RBC) lysis buffer. Cells were then incubated with ~1.6 × 10⁶ anti-EpCAM antibody-coated magnetic beads. Cancer cells adhering to beads were retrieved by running the isolation protocol on the IsoFlux™ machine (Fluxion, Alameda, CA, USA). Isolated cancer cells were fixed in 4% PFA and added onto glass slides, on which a circle with the same size as the magnet had been drawn using a water-repellent Dako pen. The glass slide was placed on top of the magnet when adding or removing buffer from the cells. The cells were stained with PE conjugated anti-CD45 [5B-1] antibody (130-080-201; MACS MiltenyiBiotec, San Diego, CA, USA; 1:100), FITC-conjugated anti-CK [CK3-6H5] antibody (130-080-101; MACS MiltenyiBiotec, 1:10), and pDAPK (orb156534; BiorByt, St. Louis, MO, USA). The cells were stained with DAPI to enable the visualization of cell nuclei. The sample was mounted using Dako Faramount aqueous mounting medium.

Isolated cells were mounted and fixed on slides and were blocked for 5 min in 10% normal donkey serum (NDS). Hereafter cells were then stained with PE-conjugated anti-CD45 [5B-1] antibody (1:200; MACS Miltenyi Biotec). Cells were then permeabilized using 0.2% Triton X-100 in PBS containing 0.5% BSA and 2 mM EDTA followed by staining of the cells with FITC-conjugated anti-Cytokeratin [CK3-6H5] antibody (1:10; MACS Miltenyi Biotec). To stain the cell nuclei, cells were incubated in DAPI afterwards. The sample was mounted with Dako Faramount Aqueous Mounting Medium. Images were captured with a fluorescent microscope (Axio Scan.Z1, Zeiss). CTCs are defined as CK-positive, CD45-negative, and nucleated cells.

Isolated cells were mounted and fixed on slides and inhibited for 5 min in 10% normal donkey serum and subsequently stained with PE-conjugated anti-CD45 (5B-1) antibody (1:200; MACS Miltenyi Biotec, Bergisch Gladbach, Germany). Then, they were permeabilized using 0.2% Triton X-100 in PBS containing 0.5% BSA and 2 mM EDTA and stained with FITC-conjugated anti-cytokeratin (CK3-6H5) antibody (1:10; MACS Miltenyi Biotec, Bergisch Gladbach, Germany) and Alexa Fluor^® 647-conjugated anti-PD-L1 antibody [SP142] (1:100; Abcam, New Taipei City, Taiwan). To stain the cell nuclei, cells were incubated in DAPI afterwards. For mounting, Dako Faramount Aqueous Mounting Medium (Agilent, Santa Clara, CA, USA) was used. Images were captured using a fluorescent microscope (Axio Scan.Z1, Zeiss, Oberkochen, Germany). CTCs were categorized into CK-positive, CD45-negative, and nucleated.