TY - JOUR
T1 - Myeloid-derived suppressor cell measurements in fresh and cryopreserved blood samples
AU - Kotsakis, Athanasios
AU - Harasymczuk, Malgorzata
AU - Schilling, Bastian
AU - Georgoulias, Vasilis
AU - Argiris, Athanassios
AU - Whiteside, Theresa L.
N1 - Funding Information:
Supported in part by the NIH grant PO-1 CA109688 to TLW. A. Kotsakis is supported by a scholarship from the Hellenic Society of Medical Oncology .
Copyright:
Copyright 2012 Elsevier B.V., All rights reserved.
PY - 2012/7/31
Y1 - 2012/7/31
N2 - Myeloid-derived suppressor cells (MDSC) present in the human peripheral blood, represent a heterogeneous population of cells with monocytic and granulocytic features. To provide guidelines for reliable assessments of the frequency and function of MDSC, we compared fresh vs. cryopreserved peripheral blood mononuclear cell (PBMC) samples obtained from normal controls and patients with cancer. PBMC were obtained from 4 healthy donors and 21 patients with cancer. They were stained with labeled antibodies, and the frequency of DR -/LIN -/CD11b +, DR -/LIN -/CD15 +, DR -/LIN -/CD33 + and DR -/low/CD14 + cells was determined by flow cytometry before and after cryopreservation. CFSE-based suppressor assays were used to test inhibitory functions of MDSC. Arginase I expression and reactive oxygen species (ROS) upregulation in MDSC subsets were evaluated by flow cytometry. The DR -/low/CD14 + and DR -/LIN -/CD11b + subsets of MDSC were found to be more resistant to the cryopreservation/thawing procedure compared to the DR -/LIN -/CD15 + and DR -/LIN -/CD33 + subsets. The frequency of the latter two MDSC subsets was significantly reduced after cryopreservation. All but DR -/LIN -/CD15 + cells inhibited proliferation of autologous CSFE-labeled CD4 + cells but lost suppressor activity after cryopreservation. Only DR -/LIN -/CD15 + cells were positive for Arginase I, but lost its expression after cryopreservation. Only fresh DR -/LIN -/CD11b + and DR -/LIN -/CD15 + cells produced ROS after in vitro stimulation.Studies of human MDSC should be performed in fresh blood samples. If samples have to be cryopreserved, monitoring of CD11b + and CD14 + MDSC subsets provides the most reliable results. Arginase I expression or stimulated ROS production assessed by flow cytometry are useful markers for MDSC subsets only in fresh samples.
AB - Myeloid-derived suppressor cells (MDSC) present in the human peripheral blood, represent a heterogeneous population of cells with monocytic and granulocytic features. To provide guidelines for reliable assessments of the frequency and function of MDSC, we compared fresh vs. cryopreserved peripheral blood mononuclear cell (PBMC) samples obtained from normal controls and patients with cancer. PBMC were obtained from 4 healthy donors and 21 patients with cancer. They were stained with labeled antibodies, and the frequency of DR -/LIN -/CD11b +, DR -/LIN -/CD15 +, DR -/LIN -/CD33 + and DR -/low/CD14 + cells was determined by flow cytometry before and after cryopreservation. CFSE-based suppressor assays were used to test inhibitory functions of MDSC. Arginase I expression and reactive oxygen species (ROS) upregulation in MDSC subsets were evaluated by flow cytometry. The DR -/low/CD14 + and DR -/LIN -/CD11b + subsets of MDSC were found to be more resistant to the cryopreservation/thawing procedure compared to the DR -/LIN -/CD15 + and DR -/LIN -/CD33 + subsets. The frequency of the latter two MDSC subsets was significantly reduced after cryopreservation. All but DR -/LIN -/CD15 + cells inhibited proliferation of autologous CSFE-labeled CD4 + cells but lost suppressor activity after cryopreservation. Only DR -/LIN -/CD15 + cells were positive for Arginase I, but lost its expression after cryopreservation. Only fresh DR -/LIN -/CD11b + and DR -/LIN -/CD15 + cells produced ROS after in vitro stimulation.Studies of human MDSC should be performed in fresh blood samples. If samples have to be cryopreserved, monitoring of CD11b + and CD14 + MDSC subsets provides the most reliable results. Arginase I expression or stimulated ROS production assessed by flow cytometry are useful markers for MDSC subsets only in fresh samples.
KW - Cell cryopreservation
KW - Flow cytometry
KW - MDSC subsets
KW - Myeloid-derived suppressor cells (MDSC)
UR - http://www.scopus.com/inward/record.url?scp=84861572331&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84861572331&partnerID=8YFLogxK
U2 - 10.1016/j.jim.2012.04.004
DO - 10.1016/j.jim.2012.04.004
M3 - Article
C2 - 22522114
AN - SCOPUS:84861572331
VL - 381
SP - 14
EP - 22
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -