Immunological Investigations, 2013; 42(4): 369–381 Copyright © Informa Healthcare USA, Inc. ISSN: 0882-0139 print / 1532-4311 online DOI: 10.3109/08820139.2013.773337 Measurement of Suppressor Activity of T CD4+CD25+ T Reg Cells Using Bromodeoxyuridine Incorporation Assay Claudia E. A ´ valos-Martı ´nez, 1 Juan Carlos Rodrı ´guez-Alba, 2,4 Laura Berro ´ n-Ruiz, 2,3 Hector Romero-Ramı ´rez, 2 Leopoldo Santos-Argumedo, 2 Luis Antonio Jime ´ nez-Zamudio, 1 Maria Lilia Domı ´nguez-Lo ´ pez, 1 Armando Vega-Lo ´ pez, 5 and Ethel Garcı ´a-Latorre 1 1 Laboratorio de Inmunoquímica I, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Miguel Hidalgo, Mexico 2 Departamento de Biomedicina Molecular, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Zacatenco, Mexico 3 Unidad de Investigación en Inmunodeficiencias, Instituto Nacional de Pediatría, San Pedro Zacatenco, Coyoacán, D.F. Mexico 4 Departamento de Biomedicina. Instituto de Ciencias de la Salud, Universidad Veracruzana, Jalapa, Veracruz 5 Laboratorio de Toxicología Ambiental, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Zacatenco, Mexico The suppressor effect of T regulatory lymphocytes in co-cultures with T effector cells obtained by magnetic columns from healthy donors and activated by CD3/CD28 was measured by a proliferation assay using BrdU incorporation and an ELISA test. Tritiated thymidine incorporation was used as a reference since it is the gold standard for proliferation assays. Both methods were used simultaneously in the same samples in order to compare them. Correlation between them was statistically significant (p < 0.001). The purification using magnetic columns was very efficient since CD4þCD25þ cells were also FOXP3þ therefore; they were identified as suppressor T cells. The use of BrdU incorporation in suppression assays is an excellent method that avoids the use of radioactive contaminating materials. Keywords Regulatory T cells, Effector T cells, Thymidine incorporation, BrdU incor- poration, Suppression by T reg cell Address correspondence to Prof. Ethel García-Latorre, Laboratorio de Inmunoquímica I, Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala s/n, Colonia Santo Tomás, México D.F. 11340, México. E-mail: ethelagarcia@hotmail.com Immunol Invest Downloaded from informahealthcare.com by 201.141.242.15 on 04/05/13 For personal use only.