Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Four color compensation.

C C Stewart1, S J Stewart

  • 1Laboratory of Flow Cytometry, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. Stewart@sc3101.med.buffalo.edu

Cytometry
|August 10, 1999
PubMed
Summary
This summary is machine-generated.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Soft Tissue Sarcomas in Octogenarian Patients: Are Treatment Options and Oncological Outcomes Different? A SEER Retrospective Study.

Clinical oncology (Royal College of Radiologists (Great Britain))·2023
Same author

Design of magnetic hybrid nanostructured lipid carriers containing 1,8-cineole as delivery systems for anticancer drugs: Physicochemical and cytotoxic studies.

Colloids and surfaces. B, Biointerfaces·2021
Same author

On the deviation from a Curie-Weiss behavior of the ZnFe<sub>2</sub>O<sub>4</sub> susceptibility: A combined ab-initio and Monte-Carlo approach.

Heliyon·2019
Same author

First-in-human phase 1 study of margetuximab (MGAH22), an Fc-modified chimeric monoclonal antibody, in patients with HER2-positive advanced solid tumors.

Annals of oncology : official journal of the European Society for Medical Oncology·2017
Same author

Large-scale partial purification of phytochrome from green leaves of Avena sativa L.

Planta·2013
Same author

Monoclonal antibodies directed to phytochrome from green leaves of Avena sativa L. cross-react weakly or not at all with the phytochrome that is most abundant in etiolated shoots of the same species.

Planta·2013
Same journal

Measurement of the distribution of red blood cell deformability using an automated rheoscope.

Cytometry·2002
Same journal

Flow cytometric-based isolation of nucleated erythroid cells during maturation: an approach to cell surface antigen studies.

Cytometry·2002
Same journal

Flow cytometric immunophenotyping test for staging/monitoring neuroblastoma patients.

Cytometry·2002
Same journal

Determination of bronchoalveolar lavage leukocyte populations by flow cytometry in patients investigated for respiratory disease.

Cytometry·2002
Same journal

Multicenter clinical experience with flow cytometric method for fetomaternal hemorrhage detection.

Cytometry·2002
Same journal

Separation of live cells in different phases of the cell cycle for gene expression analysis.

Cytometry·2002
See all related articles

Flow cytometry compensation is challenging due to tandem dye variability. Unique compensation is required for each tandem antibody, impacting data accuracy and necessitating software solutions for reliable immunophenotyping.

Area of Science:

  • Immunophenotyping
  • Flow Cytometry
  • Fluorescence Spectroscopy

Background:

  • Four-color immunophenotyping is routinely performed using single or dual laser flow cytometers.
  • Common fluorochromes include FITC, PE, PE-TR, and PE-CY5 (or PerCP) for single lasers, with APC replacing PE-TR in dual-laser systems.
  • Tandem dyes containing PE affect multiple detectors, leading to compensation complexities.

Purpose of the Study:

  • To investigate the variability in compensation requirements for tandem conjugated antibodies in flow cytometry.
  • To identify the causes of inconsistent compensation and its impact on data accuracy.
  • To evaluate potential solutions for addressing tandem dye compensation challenges.

Main Methods:

  • Analysis of compensation requirements for various tandem conjugated antibodies from different batches and suppliers.

Related Experiment Videos

  • Assessment of the impact of improper compensation on fluorescence detection, particularly for PE-CY5.
  • Evaluation of single labeled particles and dual/triple labeled cells for compensation verification.

    • Main Results:

    • Each tandem conjugated antibody, regardless of supplier or batch, requires unique compensation.
    • Inconsistent compensation leads to erroneous data and compromises the utility of single labeled particles.
    • Improper compensation can significantly reduce or eliminate the detection of fluorescence from PE-CY5 conjugates.
    • Variations in energy transfer between PE and TR/CY5, due to uncontrolled conjugation chemistry, cause these compensation issues.

    Conclusions:

    • The inherent variability in tandem dye chemistry necessitates unique compensation for each antibody conjugate.
    • Software-based compensation offers a practical solution by enabling the creation of unique compensation matrices for each antibody tandem conjugate.
    • Standardized conjugation protocols or alternative fluorochromes may be needed to improve compensation consistency in multicolor flow cytometry.