Dataset Information

Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.

ABSTRACT: Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.

SUBMITTER: Finak G

PROVIDER: S-EPMC4748244 | biostudies-other | 2016 Feb

REPOSITORIES: biostudies-other

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Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.

Finak Greg G Langweiler Marc M Jaimes Maria M Malek Mehrnoush M Taghiyar Jafar J Korin Yael Y Raddassi Khadir K Devine Lesley L Obermoser Gerlinde G Pekalski Marcin L ML Pontikos Nikolas N Diaz Alain A Heck Susanne S Villanova Federica F Terrazzini Nadia N Kern Florian F Qian Yu Y Stanton Rick R Wang Kui K Brandes Aaron A Ramey John J Aghaeepour Nima N Mosmann Tim T Scheuermann Richard H RH Reed Elaine E Palucka Karolina K Pascual Virginia V Blomberg Bonnie B BB Nestle Frank F Nussenblatt Robert B RB Brinkman Ryan Remy RR Gottardo Raphael R Maecker Holden H McCoy J Philip JP

Scientific reports 20160210

Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratorie ...[more]

PMID: 26861911

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Project description:BackgroundPersisters and viable but non-culturable (VBNC) cells are two phenotypic variants known to be highly tolerant to antibiotics. Although both cell types are stained as live and often appear as nongrowing during antibiotic treatment, the only distinguishing feature is the ability of persisters to recolonize in standard culture media in the absence of antibiotics. Despite considerable progress in the characterization of persister formation mechanisms, their resuscitation mechanisms remain unclear due to technical limitations in detecting and isolating these cell types in culture environments that are highly heterogeneous.ResultsIn this study, we used a methodology integrating flow cytometry, fluorescent protein expression systems and ampicillin-mediated cell lysing technique to monitor persister resuscitation at the single-cell level. With this method, we were able to investigate the effects of various culture conditions (e.g., antibiotic treatment time, the length of the stationary phase in overnight pre-cultures, or pretreatment of cells with a metabolic inhibitor) on persister resuscitation. Although we observed long-term pre-cultures have many more VBNC cells compared to short-term pre-cultures, only a small fraction of non-lysed cells was able to resuscitate in all conditions tested. Regardless of pre-culturing and ampicillin treatment times, these persister cells started to resuscitate within 1 hour, after they were transferred to fresh liquid media, with the same doubling time that normal cells have. Our analysis further showed that ampicillin was not able to lyse the cells in the presence of arsenate, a metabolic inhibitor commonly used to increase bacterial persistence. However, the removal of arsenate during antibiotic treatment resulted in cell lysis and a reduction in persister levels despite the significant decrease in ATP levels in the cells.ConclusionsThe strategy presented in this study helps us monitor persister resuscitation at the single-cell level, and simultaneously quantify persister, VBNC and dead cell subpopulations in ampicillin-treated cultures. Our results indicate that the characterization of persister resuscitation with flow cytometry will enhance the current molecular-level understanding of persistence and its evolution.

Dataset Information

Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.

Publications

Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.

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