Multiparametric Flow Cytometry Analysis

After washing the PBMCs in FACS buffer (phosphate-buffered saline, 1% fetal bovine serum, and 0.5 mmol/l ethylenediaminetetraacetic acid), the following antibodies were used for flow cytometry: VioBlue-conjugated anti-human CD4 (clone VIT4; Miltenyi Biotec, Bergisch Gladbach, Germany), FITC-conjugated anti-human CD8 (clone RPA-T8; BD Biosciences, Heidelberg, Germany) and CD25 (clone B1.49.9; Beckman Coulter, Marseille, France), APC-conjugated anti-human PD-1 (clone EH12.2H7; Biolegend, San Diego, CA) and CD45RA (clone HI100; Biolegend), and PE-conjugated anti-human Tim-3 (clone F38-2E2; Biolegend). After staining, the cells were washed in FACS buffer and analyzed using a MACSQuant flow cytometer with MACSQuantify software (Miltenyi Biotec). In this study, the percentages of PD-1+ and Tim-3+ T cells were calculated as percentages of the total CD4+ or CD8+ T cells. Tregs were identified as CD4+ CD45RA- CD25high cells [26 (link)] and were calculated as a percentage of the CD4+ lymphocytes. MDSCs were identified as CD11b + CD33+ cells [27 (link)] and were calculated as a percentage of the total PBMCs.

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Shindo Y., Hazama S., Suzuki N., Iguchi H., Uesugi K., Tanaka H., Aruga A., Hatori T., Ishizaki H., Umeda Y., Fujiwara T., Ikemoto T., Shimada M., Yoshimatsu K., Takenouchi H., Matsui H., Kanekiyo S., Iida M., Koki Y., Arima H., Furukawa H., Ueno T., Yoshino S., Fujita T., Kawakami Y., Nakamura Y., Oka M, & Nagano H. (2017). Predictive biomarkers for the efficacy of peptide vaccine treatment: based on the results of a phase II study on advanced pancreatic cancer. Journal of Experimental & Clinical Cancer Research : CR, 36, 36.

Publication 2017

CD45RA (clone HI100; MACSQuant flow cytometer MACSQuantify software

Antibodies Buffer Cd11b Cd25 Cd4 lymphocytes Cd8 t cells Cells Clone Ethylenediaminetetraacetic acid Fetal bovine serum Fitc Flow cytometry Human Mdscs Phosphate Saline T cells Tim 3

Corresponding Organization :

Other organizations : Yamaguchi University, Shikoku Cancer Center, Osaka City University, Tokyo Women's Medical University, University of Miyazaki, Okayama University, Tokushima University, Tokyo Women's Medical University Adachi Medical Center, Yamaguchi University Hospital, Keio University, University of Chicago

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Variable analysis

independent variables

Antibodies used for flow cytometry: VioBlue-conjugated anti-human CD4, FITC-conjugated anti-human CD8 and CD25, APC-conjugated anti-human PD-1 and CD45RA, and PE-conjugated anti-human Tim-3

dependent variables

Percentages of PD-1+ and Tim-3+ T cells calculated as percentages of the total CD4+ or CD8+ T cells
Tregs identified as CD4+ CD45RA- CD25high cells and calculated as a percentage of the CD4+ lymphocytes
MDSCs identified as CD11b + CD33+ cells and calculated as a percentage of the total PBMCs

control variables

Cells were washed in FACS buffer (phosphate-buffered saline, 1% fetal bovine serum, and 0.5 mmol/l ethylenediaminetetraacetic acid) before staining and analysis

controls

No positive or negative controls were explicitly mentioned in the provided information

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