Single-cell Isolation and Flow Cytometry Analysis

A single-cell suspension of co-culture cells and grafted corneas was prepared as previously described^{7 (link),8 (link),48 (link)}. To avoid non-specific staining, the cells were blocked with an anti-FcR-blocking antibody (eBioscience, San Diego, CA, USA). The isolated cells were stained with the following antibodies: FITC-anti-CD4 (RM4-5), and anti-IFN-γ (XMG1.2) antibodies (BioLegend, San Diego, CA, USA). The stained cells were examined using flow cytometry and the data were analyzed using FlowJo software X 10.5.3 (FlowJo LLC, Ashland, OR, USA).

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Zhu J., Inomata T., Nakamura M., Fujimoto K., Akasaki Y., Fujio K., Yanagawa A., Uchida K., Sung J., Negishi N., Nagino K., Okumura Y., Miura M., Shokirova H., Kuwahara M., Hirosawa K., Midorikawa-Inomata A., Eguchi A., Huang T., Yagita H., Habu S., Okumura K, & Murakami A. (2022). Anti-CD80/86 antibodies inhibit inflammatory reaction and improve graft survival in a high-risk murine corneal transplantation rejection model. Scientific Reports, 12, 4853.

Publication 2022

anti-FcR-blocking antibody FlowJo software X 10.5.3

Antibodies Cells Co culture Corneas Fitc Flow cytometry Ifn γ

Corresponding Organization :

Other organizations : Juntendo University, Juntendo University Hospital

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Variable analysis

independent variables

Co-culture cells and grafted corneas

dependent variables

CD4+ T cells
IFN-γ expression

control variables

Anti-FcR-blocking antibody for non-specific staining

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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