Phenotypic Analysis of B Cells and Tregs in Donor Expansion

For “donor” B cell expansion experiments, flow cytometry was performed on cultured PBMC on indicated days using antibodies against CD19-PC7, CD80-PE, CD86-PC5, HLA-DR-ECD (all from Beckman-Coulter, Miami, FL). To phenotype Tregs, antibodies against CD4-FITC, CD127-PE, CD3-ECD, CD25-PC7 (all from Beckman-Coulter) and FOXP3-PC5 (eBioscience, San Diego, CA) were used on days 0, 14 and 21, (also on day 28 in longer-term cultures). All detection was performed on a Beckman-Coulter FC500 flow cytometer as previously described^{9 ,24 (link),25 (link)}. The gating strategy used for the analyses including that for the negative controls is shown in Supplemental Fig. 1.

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Mathew J.M., Voss J.H., McEwen S.T., Konieczna I., Chakraborty A., Huang X., He J., Gallon L., Kornbluth R.S, & Leventhal J.R. (2018). Generation and Characterization of Alloantigen-Specific Regulatory T Cells For Clinical Transplant Tolerance. Scientific Reports, 8, 1136.

Publication 2018

CD19-PC7 CD80-PE HLA-DR-ECD CD4-FITC CD127-PE CD3-ECD CD25-PC7 FOXP3-PC5 FC500 flow cytometer

Antibodies B cell Cd25 Donor Fitc Flow cytometry Hla dr Phenotype

Corresponding Organization : Northwestern University

Other organizations : Multimeric Biotherapeutics (United States)

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Variable analysis

independent variables

Days of culture (0, 14, 21, 28)

dependent variables

Percentage of CD19+ B cells expressing CD80, CD86, HLA-DR
Percentage of CD4+CD25+FOXP3+ regulatory T cells

control variables

Antibodies used for flow cytometry (CD19-PC7, CD80-PE, CD86-PC5, HLA-DR-ECD, CD4-FITC, CD127-PE, CD3-ECD, CD25-PC7, FOXP3-PC5)
Flow cytometer (Beckman-Coulter FC500)
Cell type (PBMC)

positive controls

None specified

negative controls

Negative controls for flow cytometry analysis as shown in Supplemental Fig. 1

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