Longitudinal Lymphocyte Phenotyping Analysis

Absolute lymphocyte count (ALC) and lymphocyte phenotype were evaluated weekly for 4 weeks, monthly for 12 months, and every 6 months thereafter, as previously described [30 (link)]. The fluorochrome labeled monoclonal antibodies anti-CD3-Alexa 700, anti-CD3-PerCP, anti-CD4-V450, anti-CD4-PE, anti-CD8-APC Fluor780, anti-CD8-PacBlue, anti-CD16-FITC, anti-CD20 PECy7, anti-CD45-PerCP, anti-CD45RA-APC, anti-CD56-APC, anti-Ki67-FITC and anti-CD197 PECy7 (BD Biosciences, Franklin Lakes, NJ), and anti-CD45RA-QDOT655 (Invitrogen, Carlsbad, CA) were used. ALC was determined using Trucount beads (BD Biosciences). Cells were interrogated using an LRSII Flow Cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, San Carlos, CA). Memory and regulatory phenotypes were defined as previously described [31 (link)–34 (link)].

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Kirk A.D., Guasch A., Xu H., Cheeseman J., Mead S.I., Ghali A., Mehta A.K., Wu D., Gebel H., Bray R., Horan J., Kean L.S., Larsen C.P, & Pearson T.C. (2014). Renal Transplantation Using Belatacept Without Maintenance Steroids or Calcineurin Inhibitors. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 14(5), 1142-1151.

Publication 2014

anti-CD3-Alexa 700 anti-CD3-PerCP anti-CD4-V450 anti-CD4-PE anti-CD8-APC Fluor780 anti-CD8-PacBlue anti-CD16-FITC anti-CD20 PECy7 anti-CD45-PerCP anti-CD45RA-APC anti-CD56-APC anti-Ki67-FITC anti-CD197 PECy7 anti-CD45RA-QDOT655 Trucount beads LRSII Flow Cytometer

Anti cd3 Cells Fitc Fluorochrome Lymphocyte Lymphocyte count Memory Monoclonal antibodies Phenotypes Tree

Corresponding Organization : Emory University

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Variable analysis

independent variables

Time (weekly for 4 weeks, monthly for 12 months, and every 6 months thereafter)

dependent variables

Absolute lymphocyte count (ALC)
Lymphocyte phenotype (including CD3, CD4, CD8, CD16, CD20, CD45, CD45RA, CD56, Ki67, CD197)

control variables

Fluorochrome labeled monoclonal antibodies (anti-CD3-Alexa 700, anti-CD3-PerCP, anti-CD4-V450, anti-CD4-PE, anti-CD8-APC Fluor780, anti-CD8-PacBlue, anti-CD16-FITC, anti-CD20 PECy7, anti-CD45-PerCP, anti-CD45RA-APC, anti-CD56-APC, anti-Ki67-FITC and anti-CD197 PECy7 from BD Biosciences, and anti-CD45RA-QDOT655 from Invitrogen)
Trucount beads (BD Biosciences) for ALC determination
LRSII Flow Cytometer (BD Biosciences) for cell interrogation
FlowJo software (Tree Star, San Carlos, CA) for data analysis
Memory and regulatory phenotypes defined as previously described [31–34]

controls

No explicit positive or negative controls were mentioned.

Annotations

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