Multiparametric Flow Cytometry Analysis

Single-cell suspension was obtained as previously described [25 (link)]. The cells were stained with following antibodies; APC-labeled anti-human PD-L1 antibody (#329707, clone 29E.2A3, BioLegend, San Diego, CA, USA), APC-labeled anti-mouse PD-L1 antibody (#124311, clone 10F.9G2, BioLegend), FITC-labeled anti-CD45 (#103107, clone 30-F11, BioLegend), monoclonal PerCP/Cyanine5.5-labeled anti-CD31 (#102419, clone 390, BioLegend), monoclonal PE-labeled anti-CD90.2 (#105307, clone 30-H12, BioLegend), human IgG isotype control antibody (#400322, clone MPC-11, BioLegend), and murine-IgG isotype control antibody (#400612, clone RTK4530, BioLegend). Red blood cell lysis buffer (420302, BioLegend) and Debris Removal Solution (130-109-398, Miltenyi Biotec, Bergisch Gladbach, Germany) were also used. Dead cells (1:1000 dilution) were stained using a Zombie NIR Fixable Viability Kit (423106, BioLegend). Stained cells were analyzed using flow cytometry (FACSLyric; BD Biosciences, Franklin Lakes, NJ, USA), and data were analyzed using the FlowJo software (BD Biosciences).

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Kawasaki K., Noma K., Kato T., Ohara T., Tanabe S., Takeda Y., Matsumoto H., Nishimura S., Kunitomo T., Akai M., Kobayashi T., Nishiwaki N., Kashima H., Maeda N., Kikuchi S., Tazawa H., Shirakawa Y, & Fujiwara T. (2023). PD-L1-expressing cancer-associated fibroblasts induce tumor immunosuppression and contribute to poor clinical outcome in esophageal cancer. Cancer Immunology, Immunotherapy, 72(11), 3787-3802.

Publication 2023

APC-labeled anti-mouse PD-L1 antibody FITC-labeled anti-CD45 Red blood cell lysis buffer Debris Removal Solution Zombie NIR Fixable Viability Kit FACSLyric

Anti antibody Antibodies Buffer Cd90 Cells Clone Dilution Fitc Flow cytometry Human Igg antibody Isotype Murine Pd l1 Red blood cell

Corresponding Organization :

Other organizations : Okayama University, Okayama University Hospital

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Variable analysis

independent variables

Antibodies used for staining the cells
Lysis buffer and Debris Removal Solution used

dependent variables

PD-L1 expression on cells
CD45, CD31, and CD90.2 expression on cells
Viability of cells

control variables

Single-cell suspension preparation method (as previously described in reference 25)
Flow cytometry (FACSLyric; BD Biosciences) for analyzing the stained cells
FlowJo software (BD Biosciences) for data analysis

controls

Positive controls: Human IgG isotype control antibody, Murine-IgG isotype control antibody
Negative control: Zombie NIR Fixable Viability Kit for staining dead cells

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