Validation of Manufacturers Recommended Working Volumes of Fluorochrome-conjugated Monoclonal Antibodies for Flowcytometric Analysis of Lymphocytes

Abstract

Background: Flow cytometry is a robust and rapidly growing technique used to analyse multiple parameters concurrently on a single cell basis. Cell populations can be characterised using a combination of both surface and intracellular antigens. The act of generating the best research data under given circumstances begins with a well-designed reagent optimisation protocol. Applying flow cytometric analysis to obtain reliable and dependable research data requires establishing the best working volumes of the monoclonal antibodies. Aim: This study aimed to provide practical illustrations on the approach for determining optimal working volumes (concentrations) of fluorochrome-conjugated monoclonal antibodies for flow cytometry. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were stained using three different volumes of respective fluorochrome-conjugated monoclonal antibodies: one volume below, a volume above and the volume recommended by the manufacturers. The antibodies analysed include CD3-FITC, CD4-APC-Cy7, CD19-Alexa Fluor 700, CD45-AmCyan, CD28-PE, and CD45-AmCyan isotype control.  Depending on the availability of cells, a total of 10,000 to 30,000 events were acquired for analysis. Combinations of the mean fluorescence intensity (MFI), the number of events in the population of interest, and the clearance of the Isotype control histogram peak from the positive population were used to determine the best working volumes of the mAbs used. Results: The study reported optimum working volumes of 10µL for CD45-Amcyn, 20µL for CD3-FITC, 10µL for CD4-APC-Cy7, 10µL for CD19-Alexa Fluor 700, 20µL for CD28-PE. We confirmed the recommended volumes provided by the manufacturer for CD3-FITC and CD28-PE. However, higher volumes of CD45-Amcyn, CD4-APC-Cy7 and CD19-Alexa Fluor 700 were found more optimal than the recommended volumes supplied by manufacturers. Conclusion: The application of a simple validation experiment for the working volumes (concentrations) of fluorochrome-conjugated monoclonal antibodies, like the one described here, is recommended as an integral part of the optimisation protocol for flow cytometry.