Apc cyanine7 mouse anti human cd45 2d1

Cells enriched with RosetteSep were fixed with 4% paraformaldehyde and blocked using a blocking antibody diluent (PerkinElmer/Akoya Biosciences, San Diego, CA, USA). The cells were further incubated with an optimal dilution of APC/Cyanine7 mouse anti-human CD45 (2D1) (BioLegend, San Diego, CA, USA), PE mouse anti-human pan-cytokeratin (C-11) (Cayman Chemical, Ann Arbor, MI, USA), or isotype-matched control mouse IgG (BD Biosciences, San Jose, CA, USA). The antibodies used are listed in Table 2. The nuclei were counterstained with 4’-6-diamidino-2-phenylindole (DAPI) (PerkinElmer/Akoya Biosciences). Immunofluorescence was detected using a fluorescence microscope (BZ-X800; Keyence, Itasca, IL, USA).

After separation of blood using Rosettesep, enriched cells were labeled with fluorescent dye-conjugated antibodies, APC/Cyanine7 mouse anti-human CD45 (2D1) (BioLegend, San Diego, CA, USA), PE mouse anti-human pan-cytokeratin (cytokeratin 4, 5, 6, 8, 10, 13, and 18) (C-11) (Cayman Chemical), and BV421 mouse anti-human PD-L1 (29E.2A3) (BD Biosciences). Cell pellets were resuspended in 500 μL PBS and counted through multiparametric flow cytometry using a BD FACSAriaII (BD Biosciences). CD45 negative and Pan-CK positive cells were defined as CTCs. The data were analyzed using FlowJo version 10.9.0 (BD Biosciences). Isotype-matched control IgGs for individual mouse monoclonal antibodies (BD Biosciences) and CompBeads anti-mouse Ig, κ/Negative Control Compensation Particles Set (BD Biosciences) were used for compensation. The antibodies used are listed in Table 2. To detect dead cells, 7-aminoactinomycin D (7-AAD; BD Biosciences) was added to the buffer immediately before flow cytometry. In this study, we considered the 7-AAD(−)CD45(−)PanCK(+) populations detected by flow cytometry as CTCs.