Validation of Manufacturers Recommended Working Volumes of Fluorochrome-conjugated Monoclonal Antibodies for Flowcytometric Analysis of Lymphocytes
Asian Hematology Research Journal,
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.
- Fluorochrome conjugated
- flow cytometry
- working volume
- monoclonal antibodies
How to Cite
Brown M, Wittwer C.Flow cytometry: principles and clinical applications in hematology. Clinical chemistry, 2000; 46(8):1221-1229.
Huh D, et al. Microfluidics for flow cytometric analysis of cells and particles. Physiological measurement. 2005;26(3): R73.
Wang L, Hoffman RA. Flow cytometer performance characterization, standardization and control, in Single Cell Analysis. Springer. 2017;171-199.
Adan A, et al. Flow cytometry: basic principles and applications. Critical reviews in biotechnology. 2017;37(2):163-176.
Tarafder S, Khanom KH, Sattar H. Flow cytometric immunophenotyping including Bcl-2 detection on fine needle aspirates of lymph node in the diagnosis of Diffuse Large B-Cell Lymphoma. chemotherapy. 2018;5:6.
Fazio F, et al. Flow cytometry and automatic blood cell analysis in striped bass Morone saxatilis (Walbaum, 1792): A new hematological approach. Aquaculture. 2019;513:734398.
Craig FE and KA Foon, Flow cytometric immunophenotyping for hematologic neoplasms. Blood. 2008;111(8):3941-3967.
Wlodkowic D, et al. Apoptosis and beyond: cytometry in studies of programmed cell death, in Methods in cell biology. Elsevier. 2011;55-98.
Lin P, et al. Flow cytometric immunophenotypic analysis of 306 cases of multiple myeloma. American journal of clinical pathology, 2004;121(4):482-488.
Pozarowski P, Darzynkiewicz Z. Analysis of cell cycle by flow cytometry, in Checkpoint Controls and Cancer. Springer. 2004;301-311.
Pösel C, et al. Isolation and flow cytometric analysis of immune cells from the ischemic mouse brain. JoVE (Journal of Visualized Experiments). 2016;(108):e53658.
Yen-Rei AY, et al. A protocol for the comprehensive flow cytometric analysis of immune cells in normal and inflamed murine non-lymphoid tissues. PloS one. 2016;11(3):e0150606.
Givan AL. Flow cytometry: an introduction, in Flow Cytometry Protocols. Springer. 2011;1-29.
Picot J, et al. Flow cytometry: retrospective, fundamentals and recent instrumentation. Cytotechnology. 2012; 64(2):109-130.
Perfetto SP, Chattopadhyay PK, Roederer M. Seventeen-colour flow cytometry: unravelling the immune system. Nature Reviews Immunology. 2004;4(8):648-655.
Manohar SM, Shah P, Nair A. Flow cytometry: principles, applications and recent advances. Bioanalysis, 2021; 13(3):181-198.
Hughes OR, et al. A critical appraisal of factors affecting the accuracy of results obtained when using flow cytometry in stem cell investigations: where do you put your gates? Cytometry part A. 2009; 75(9):803-810.
Greve B, et al. Flow cytometry in transfusion medicine: development, strategies and applications. Transfusion Medicine and Hemotherapy. 2004; 31(3):152-161.
Borowitz MJ, et al. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry Part B: Clinical Cytometry. 2010;78(4):211-230.
Sutherland DR, Keeney M, Illingworth A. Practical guidelines for the high‐sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry Part B: Clinical Cytometry. 2012;82(4):195-208.
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