Standardizing flow cytometry: A classification system of fluorescence standards used for flow cytometry

Schwartz, A.; Marti, Gerald E.; Poon, Randy Y. C.; Gratama, Jan W.; Fernández-Repollet, Emma

doi:10.1002/(sici)1097-0320(19981001)33:2<106::aid-cyto4>3.0.co;2-h

Cited by 108 publications

(46 citation statements)

References 18 publications

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“…As in the current study, many quantitative calculations rely on standardized calibration microsphere sets for conversion of measured fluorescence intensity data to a measure of equivalent fluorophores. These calibration particles can take many forms including standard calibration microsphere sets with fluorophores directly attached to the microsphere surface (27, 49), application-specific microspheres that bind known amounts of fluorophore-labeled proteins such as mAbs (25, 29) and other distinct types of fluorescence standards (28). However, the lack of commercially available standards compatible for use with QD probes requires alternate methods.…”

Section: Discussionmentioning

confidence: 99%

Quantitative measurement of multifunctional quantum dot binding to cellular targets using flow cytometry

Smith¹,

Giorgio²

2008

Cytometry Pt A

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Semiconductor nanocrystals such as quantum dots (QDs) are a potentially powerful resource in the fields of flow cytometry and fluorescence microscopy. QD size and fluorescence characteristics offer attractive features for use in targeted delivery systems and detection by flow cytometry. While quantitative measurements of a variety of fluorescent molecules are routinely performed, fluorophores for which no calibration standards exist, such as QDs, pose a problem for quantitation in flow cytometry. Our goal was to develop a targeted nanoparticle delivery platform as well as a corresponding method to accurately and quantitatively assess the performance of this system. We synthesized surface-modified QD probes targeted to cellular surface receptors and measured the MFI of the resulting cell-probe conjugates by flow cytometry. MFI was converted to mean equivalent R-PE intensity (MEPE) using standard calibration microspheres. Known concentrations of both R-PE and QD probes were measured by fluorometry to relate R-PE and QD fluorescence. Fluorometry results were then used to translate MEPE measurements to the number of bound QD probes. The targeted probes exhibited superior binding characteristics over unmodified and untargeted particles. This binding interaction was shown to be specific and mediated by the NGR targeting peptide tethered to the QD surface. The calibration method developed to assess this system proved successful at converting raw fluorescence data to quantitative probe binding values. We demonstrate the synthesis and performance of a highly modular nanoparticle system capable of targeted binding and fluorescent imaging. The calibration method implemented to quantify the performance of this system represents a potentially powerful tool to utilize truly quantitative flow cytometry measurements with an array of fluorescent molecules, including QDs. '

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Section: Discussionmentioning

confidence: 99%

Quantitative measurement of multifunctional quantum dot binding to cellular targets using flow cytometry

Smith¹,

Giorgio²

2008

Cytometry Pt A

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“…A standard calibration plot is used to monitor the daily instrument performance [16]. This quality control (QC) plot provides the user with a much higher degree of confidence in the results of their assays because it indicates not only the linearity and sensitivity of the flow cytometer instrument but also the fluorescence intensity that the instrument covers at a particular setting.…”

Section: Standard Calibration Plotmentioning

confidence: 99%

Guidelines for organizing national external quality assessment scheme for performing CD4+ T-lymphocyte determinations in persons infected with HIV in resource-limited settings

Noulsri

Pattanapanyasat

2015

Accred Qual Assur

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CD4 testing is important for the clinical monitoring of patients with HIV/AIDS. Quality assurance is therefore an essential element to ensure that the test results are reliable, reproducible, traceable, and auditable. An external quality assessment scheme (EQAS) provides an assessment of the entire testing process, including pre-and post-analytical procedures. At present, there is an increased demand for EQAS owing to an increasing number of CD4 testing laboratories in many countries. However, participating in the existing international EQAS is extremely expensive, making its implementation in resource-limited settings difficult. An ideal alternative is to have an equally qualified local or regional CD4 EQAS, which is more practical and will have a more obvious impact on these countries. The present communication aims to establish the requirements and guidelines for implementing EQAS for CD4? T-lymphocyte determination in individuals with HIV-1 infection in resource-limited regions, particularly in sub-Saharan African countries and in some countries in Southeast Asia. Guidelines including the recruitment of CD4 testing laboratories, pre-participation EQA workshops, and management of EQA blood samples along with the evaluation of the performance data are described. An example of participants' registration form, details of EQAS blood panels, participants' report form, and EQAS performance report form are also included in the guidelines. The guidelines presented herein will lay a foundation to help improve the reliability of CD4? T-lymphocyte determination in resource-limited settings.

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“…A second technique that we employed for expression validation is quantitative flow cytometry (Q-FACS) [70,71]. As a highly specific technique, flow cytometry makes it possible to quickly, and simultaneously, measure numerous cellular physical parameters on a large number of suspension cells.…”

Section: Validation Of Expression In Tumor Cells and Membrane Localizmentioning

confidence: 99%

“…We chose to use the Quantum Simply Cellular System (Bangs Laboratories, Fishers, IN), which utilizes Type IIIC calibration standards. This particular type of standard is reported to produce the best spectra matching since the beads bind the same conjugated antibody as the cell sample [71]. Quantum Simply Cellular System kits consist of five polystyrene microbead sets, where each set is labeled with a predetermined number of ligands on each bead.…”

Section: Prostate Carcinomamentioning

confidence: 99%

RNAi Validation of Pancreatic Cancer Antigens Identified by Cell Surface Proteomics

Lee

McCaffrey

et al. 2014

Molecular Diagnostics and Treatment of Pancreatic Cancer

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Standardizing flow cytometry: A classification system of fluorescence standards used for flow cytometry

Cited by 108 publications

References 18 publications

Quantitative measurement of multifunctional quantum dot binding to cellular targets using flow cytometry

Quantitative measurement of multifunctional quantum dot binding to cellular targets using flow cytometry

Guidelines for organizing national external quality assessment scheme for performing CD4+ T-lymphocyte determinations in persons infected with HIV in resource-limited settings

RNAi Validation of Pancreatic Cancer Antigens Identified by Cell Surface Proteomics

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