Experimental error control during immune monitoring by flow cytometry of positive reactors to bovine

Abstract

Flow cytometry (FC) has become the method of choice for studying the immune response to infectious agents. The development of an extensive set of monoclonal antibodies (MAb) to bovine leukocyte differentiation molecules has now made it possible immune response characterization in bovine tuberculosis (Tbc). Before extending these studies it is essential to establish results consistency. Currently indirect labeling (IL) is used in single and multiparameter analysis. This introduces the probability of experimental error because of the multiple processing steps needed. The objective of this study was to develop and test a strategy for assuring the reliability of methods for processing peripheral blood mononuclear cells (PBMC) from Tbc positive cattle. PBMC were obtained from 10 Tbc positive cows by density gradient centrifugation (Histopaque1077). Each sample was labeled with four MAb cocktails (antiCD4-IgM/antiCD25-IgG1, antiCD4/antiCD45Ro-IgG, antiCD8-IgM/antiCD25, antiCD8/antiCD45Ro), then they were washed and labeled with antiIgG1-PE/anti-IgM-FITC cocktail. Finally PBMC were fixed, stored and 10000 events were acquired with cytometer FACS-CANTO (Becton-Dickinson). Data were analyzed with FCS Express trial version. A dot plot side light scatter (SSc) vs forward light scatter (FSc) was set to define the mononuclear cell population. Then a dot-plot FSc vs fluorescence distinguished between unlabeled and labeled cells. Finally a histogram is generated to compare geometric means of fluorescence intensity (GMFs). SAS v 9.2 calculated the paired t test between duplicate GMFs. The variation in labeling between paired samples was very low, p value of paired t test >0.05 in all samples, showing that reliable results can be obtained with minimal differences introduced during sample preparation. When in FC IL is routine, statistical comparison of results from repetitions of the same sample labeled with the same MAb would be strategic as an experimental error control.