Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Maria Letizia Giardino Torchia; Raffaello Cimbro

doi:10.3791/58955

Discriminação de sete subconjuntos de células imunes por dois-fluorocromo citometria de fluxo

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Immunology and Infection

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JoVE Journal Immunology and Infection

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Discriminação de sete subconjuntos de células imunes por dois-fluorocromo citometria de fluxo

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10:58 min

March 5, 2019

DOI: 10.3791/58955-v

Maria Letizia Giardino Torchia¹, Raffaello Cimbro²

¹Oncology Research,Medimmune, LLC, ²Division of Rheumatology,Johns Hopkins University School of Medicine

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Overview

This article presents a flow cytometric protocol for identifying various immune cell types in human peripheral blood using only two fluorochromes. This method allows for the recording of five additional markers, enhancing the analysis of complex cell populations.

Key Study Components

Area of Science

Immunology
Flow Cytometry
Cell Biology

Background

Flow cytometry is a powerful tool for analyzing cell populations.
Traditional methods often require multiple fluorochromes, limiting the number of markers that can be analyzed simultaneously.
This study aims to simplify the process while expanding the analytical capabilities.
Understanding immune cell populations is crucial for various biomedical research applications.

Purpose of Study

To develop a protocol that allows for the identification of multiple immune cell types using fewer fluorochromes.
To facilitate deeper immunophenotyping with limited resources.
To improve the efficiency of flow cytometric analysis in research settings.

Methods Used

Blood samples are diluted with PBS and layered over a density gradient medium.
Flow cytometry is performed to identify cell populations.
Two fluorochromes are utilized to label the cells.
Five additional markers are recorded to enhance analysis.

Main Results

The protocol successfully identifies CD4 + and CD8 + T cells, 纬未 T cells, B cells, NK cells, and monocytes.
Five additional markers can be analyzed without compromising the quality of data.
This method demonstrates the feasibility of using fewer fluorochromes in flow cytometry.
Results indicate improved efficiency in analyzing complex immune cell populations.

Conclusions

The developed protocol enhances flow cytometric analysis by reducing the number of required fluorochromes.
This approach allows for a more comprehensive understanding of immune cell populations.
It provides a valuable tool for researchers with limited resources or equipment.

Frequently Asked Questions

What is the main advantage of this flow cytometric protocol?

The main advantage is the ability to identify multiple immune cell types using only two fluorochromes, allowing for additional markers to be recorded.

How does this method improve immunophenotyping?

It allows for deeper analysis of complex cell populations even with instruments that have a low number of detectors.

What types of cells can be identified using this protocol?

CD4 + and CD8 + T cells, 纬未 T cells, B cells, NK cells, and monocytes can be identified.

Is this method suitable for samples with limited cell numbers?

Yes, the protocol is designed to work effectively with samples that have a limited number of cells.

What is the significance of using fewer fluorochromes?

Using fewer fluorochromes simplifies the process and reduces costs while still allowing for comprehensive analysis.

Aqui, apresentamos um protocolo de fluxo cytometric para identificar CD4⁺ e CD8⁺ T células, as células γδ T, células B, células NK e monócitos no sangue periférico humano usando apenas dois fluorochromes em vez de sete. Com esta abordagem, cinco marcadores adicionais podem ser gravadas na maioria dos citômetros.

O objetivo geral dessa metodologia é expandir-se além do limite óptico da maioria dos citómetros de fluxo, permitindo que um pesquisador grave cinco marcadores adicionais para interrogar populações celulares complexas. Usando essa técnica, é possível alcançar um imunofenofoteping profundo ao trabalhar com instrumento com um baixo número de detectores ou amostra com número limitado de células. Inicie este procedimento transferindo cuidadosamente o sangue extraído para um tubo cônico de 50 mililitros.

Diluir o sangue com uma quantidade igual de PBS sem cálcio e magnésio. Adicione 15 mililitros de gradiente de densidade ao fundo de um novo tubo cônico de 50 mililitros. Sobreponha cuidadosamente o sangue diluído em cima do meio gradiente de densidade, evitando qualquer mistura entre o gradiente de densidade médio e sangue diluído.

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