Porcine CFSE mixed lymphocyte reaction and PKH-26 cell-mediated lympholysis assays.
Mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) are widely used to assess T cell responses. A major limitation of the traditional MLR and CML assays is that they require radioisotope labeling with (3)H for MLR and (51)Cr for CML, thereby limiting their use to laboratories with the capabilities to deal safely with these materials. Recently, flow cytometry with CFSE labeling has been used to detect cell division in rodent and human assays, and flow cytometry with PKH-26 labeling has been used to study cytotoxicity in murine models. Partially inbred miniature swine provide a unique large animal preclinical model for experimental transplantation, helping to bridge the gap between rodent and clinical studies. In this study, we modified the reported CFSE and PKH-26 labeling procedures for use with porcine cells, and established that these radioactive-free MLR and CML assays are comparable to traditional radioactive CML and MLR assays for assessing immunologic responses in miniature swine. To our knowledge, this is the first report that has directly compared the traditional CML/MLR with radiation-free CML/MLR in MHC-defined swine models.


OBJECTIVE
The aim of this study is to establish non-radiolabeled CSFE and PKH-26 labeling procedures for flow cytometry based CML/MLR assays that are comparable to radioactive CML/MLR assays in preclinical large animals.
Oku
Manei
M
Frontier Science Research Center, Kagoshima University, Japan.
Okumi
Masayoshi
M
Sahara
Hisashi
H
Hirakata
Atsushi
A
Onoe
Takashi
T
Griesemer
Adam D
AD
Yamada
Kazuhiko
K
D000818:Animals; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D007959:Lymphocyte Culture Test, Mixed / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry*; D011863:Radioimmunoassay; D011868:Radioisotopes / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry*; D013552:Swine; D013556:Swine, Miniature
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C070080:PKH 26; D011868:Radioisotopes; D013388:Succinimides