Nine‐color flow cytometry for accurate measurement of T cell subsets and cytokine responses. Part I: Panel design by an empiric approach

Abstract

Polychromatic flow cytometry offers the unprecedented ability to investigate multiple antigens per cell. Unfortunately, unwanted spectral overlaps and compensation problems increase when more than four colors are used, but these problems can be minimized if staining combinations are chosen carefully. We used an empiric approach to design, test and identify six-color T cell immunophenotyping reagent panels that can be expanded to include three or more functional or other markers in the FITC, PE, and APC channels without significant spectral limitations. Thirty different six-color T cell surface antigen reagent panels were constructed to identify major T cell subsets and maturational subtypes as defined by CCR7 and CD45RA expression, while excluding monocytes, B and non-viable cells. Staining performance of each panel was compared on cryopreserved cells from a single healthy donor recorded on a multiparameter cell sorter. Ten of the thirty reagent panels offered reliable resolution of T cell major and maturational surface markers. Of these, two panels were selected that showed the least spectral overlap and resulting background increase in the FITC, PE, and APC channels. These channels were left unoccupied for inclusion of additional phenotypic or functional markers, such as cytokines. Careful reagent titration and testing of multiple candidate panels are necessary to ensure quality results in multiparametric measurements. ' 2008 International Society for Advancement of Cytometry Key terms polychromatic flow cytometry; T cell immunophenotyping; fluorochrome conjugated antibody; compensation POLYCHROMATIC flow cytometry allows for detailed measurements even with small sample sizes and has recently been advanced by the development of new instrumentation, reagents and data analysis tools. Despite these significant improvements, it can be difficult to derive meaningful results when reagent combinations are expanded to include eight or more fluorescent markers. This is because unwanted spectral overlaps and measurement errors worsen as the number of fluorochromes used to label coordinately expressed cell markers increase (1-3). Using appropriate controls, software compensation algorithms can correct spillover problems post acquisition. However, due to the increased number of spectral overlaps in polychromatic reagent combinations, even properly compensated data can exhibit unwanted spreading into other measurement channels, complicating data analysis and interpretation (3,4). For example, dimly expressed markers, such as cytokines, are difficult to measure in channels where spreading in properly compensated data increases the background in the cytokine measurement channel. Such data spread in effect masks low intensity events; a problem not usually apparent when only a few stains are used simultaneously.A variety of sources contribute to this error in compensated data and are partially corrected by newer digital electronic configurations that collect and store data

Diseño de un panel multicolor para evaluar moléculas intracelulares y de superficie mediante citometría de flujo

“…Muchos fluorocromos con diferentes espectros de excitación y emisión se encuentran disponibles para marcar desde 9 hasta 17 moléculas distintas sobre una célula (3,5,16). En esta nota técnica, se muestra un método para la construcción armónica de un panel multicolor con 11 parámetros, que permite evaluar LT CD8 + fenotípica y funcionalmente a partir de células mononucleares de sangre periférica ex vivo y cultivadas.…”

“…La evaluación del panel multicolor permite identificar los conjugados de anticuerpos que crean problemas de interferencia alterando su sensibilidad y, además, evalúa el efecto de cada uno de los anticuerpos conjugados sobre la coloración completa (6,16). La evaluación del panel multicolor incluyó la construcción progresiva del panel y la fluorescencia menos uno.…”

“…Esta nota técnica muestra un método para la construcción y evaluación de paneles multicolor para el análisis fenotípico y funcional de LT CD8 + en células ex vivo, y en respuesta a un estímulo policlonal o específico con base en metodologías previamente descritas (6,8,15,16).…”

Discovering new cell populations

“…Multiplexing single cell analysis in order to detect phenotype combinations may always lead to novel hitherto unknown phenotypes. As a few examples of many, Mittag et al found new NK-T subpopulations in humans using eightcolor quantitative microscopy (1); Seder et al (2) reported cytotoxic T-lymphocytes in humans with multiple cytokine production ability using a nine-color approach (3).…”

Infinite multidimensionality

“…Increasing complexity requires new dyes, new light sources, and a demanding experimental design (12). Now, this rule of thumb has been broken.…”