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Multiplexed detection and isolation of viable low-frequency cytokine-secreting human B cells using cytokine secretion assay and flow cytometry (CSA-Flow).

ABSTRACT: The ability to functionally characterize cytokine-secreting immune cells has broad implications in both health and a range of immune-mediated and auto-immune diseases. Low-frequency cytokine-defined immune-cell subsets can play key immune-regulatory roles, yet their detailed study is often hampered by limited clinical sample availability. Commonly used techniques including intracellular cytokine staining require cell fixation, precluding subsequent functional interrogation. The cytokine-secretion assay (CSA) can overcome this limitation, though has mostly been used for detection of relatively high-frequency, single-cytokine secreting cells. We examined how adaptation of the CSA in combination with multiparametric flow-cytometry (CSA-Flow) may enable simultaneous isolation of multiple, low-frequency, cytokine-secreting cells. Focusing on human B cells (traditionally recognized as harder to assay than T cells), we show that single-capture CSA-Flow allows for isolation of highly-purified populations of both low-frequency (IL-10⁺; GM-CSF⁺) and high-frequency (TNF⁺) cytokine-defined B cells. Simultaneous detection and isolation of up to three viable and highly-purified cytokine-secreting B-cell subpopulations is feasible, albeit with some signal loss, with fractions subsequently amenable to gene expression analysis and in vitro cell culture. This multiplexing CSA-Flow approach will be of interest in many human cellular immunology contexts aiming to functionally characterize cytokine-secreting immune cells, especially when sample volumes and cell numbers are limited.

SUBMITTER: Rezk A

PROVIDER: S-EPMC7481209 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Multiplexed detection and isolation of viable low-frequency cytokine-secreting human B cells using cytokine secretion assay and flow cytometry (CSA-Flow).

Rezk Ayman A Li Rui R Bar-Or Amit A

Scientific reports 20200909 1

The ability to functionally characterize cytokine-secreting immune cells has broad implications in both health and a range of immune-mediated and auto-immune diseases. Low-frequency cytokine-defined immune-cell subsets can play key immune-regulatory roles, yet their detailed study is often hampered by limited clinical sample availability. Commonly used techniques including intracellular cytokine staining require cell fixation, precluding subsequent functional interrogation. The cytokine-secretio ...[more]

PMID: 32908164

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Project description:BackgroundCytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online).ResultsThree sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells.ConclusionICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

Project description:Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

Dataset Information

Multiplexed detection and isolation of viable low-frequency cytokine-secreting human B cells using cytokine secretion assay and flow cytometry (CSA-Flow).

Publications

Multiplexed detection and isolation of viable low-frequency cytokine-secreting human B cells using cytokine secretion assay and flow cytometry (CSA-Flow).

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