Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners

Brian Dinh; Marten A. Hoeksema; Nathanael J. Spann; Jacob Rendler; Isidoro Cobo; Christopher K. Glass; Calvin Yeang

doi:10.3791/66794

Aislamiento y criopreservación de células mononucleares de sangre periférica humana altamente via...

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Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners

Aislamiento y criopreservación de células mononucleares de sangre periférica humana altamente viables a partir de sangre total: una guía para principiantes

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10:48 min

October 25, 2024

DOI: 10.3791/66794-v

Brian Dinh¹, Marten A. Hoeksema², Nathanael J. Spann³, Jacob Rendler¹, Isidoro Cobo^4,5, Christopher K. Glass^3,6, Calvin Yeang¹

¹Division of Cardiology, Department of Medicine,University of California San Diego, ²Department of Medical Biochemistry, Amsterdam Immunity and Infection: Inflammatory diseases; Amsterdam Cardiovascular Sciences: Atherosclerosis and Ischemic Syndrome,Amsterdam UMC location University of Amsterdam, ³Department of Cellular and Molecular Medicine,University of California San Diego, ⁴Division of Clinical Immunology & Rheumatology, Department of Medicine, Heersink School of Medicine,University of Alabama at Birmingham, ⁵Comprehensive Arthritis, Musculoskeletal, Bone and Autoimmunity Center, Heersink School of Medicine,University of Alabama at Birmingham, ⁶Department of Medicine,University of California San Diego

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Overview

This protocol provides a step-by-step guide for collecting, storing, and thawing peripheral blood mononuclear cells (PBMCs) from whole blood. It also includes methods for collecting plasma and buffy coat, which are essential for various downstream analyses.

Key Study Components

Area of Science

Cell biology
Immunology
Hematology

Background

Peripheral blood mononuclear cells (PBMCs) include lymphocytes, dendritic cells, and monocytes.
PBMCs are widely used in research for various assays.
High viability PBMCs are crucial for accurate experimental results.
Collection methods impact the quality and quantity of cells obtained.

Purpose of Study

To provide a reliable protocol for PBMC collection and storage.
To ensure high viability of cells for downstream applications.
To demonstrate the collection of additional components like plasma and buffy coat.

Methods Used

Collection of PBMCs from whole blood using density gradient centrifugation.
Preparation of materials and reagents as outlined in the protocol.
Storage and thawing procedures for PBMCs.
Collection techniques for EDTA plasma and buffy coat.

Main Results

Successful collection of approximately 12 million PBMCs per tube.
High viability rates of PBMCs suitable for downstream analyses.
Demonstrated methods for plasma and buffy coat collection.
Protocol facilitates various experimental workflows including RNA sequencing and flow cytometry.

Conclusions

This protocol provides a comprehensive guide for PBMC collection.
Ensures high-quality samples for immunological and cellular studies.
Facilitates further research in various biological assays.

Frequently Asked Questions

What are PBMCs?

PBMCs are peripheral blood mononuclear cells, which include lymphocytes, dendritic cells, and monocytes.

Why is it important to collect PBMCs?

PBMCs are essential for various assays and experiments in immunology and cell biology.

How many PBMCs can be collected from one tube?

Each density gradient centrifugation tube yields approximately 12 million PBMCs.

What is the purpose of collecting plasma and buffy coat?

Plasma and buffy coat are important for additional analyses and can provide further insights into immune responses.

What downstream applications can PBMCs be used for?

PBMCs can be used for RNA sequencing, flow cytometry, and various other assays.

How do you ensure high viability of PBMCs?

Following the protocol carefully and using appropriate storage methods helps maintain high viability.

Este protocolo presenta una guía accesible para recolectar, almacenar y descongelar células mononucleares de sangre periférica adecuada para análisis posteriores y flujos de trabajo como la citometría de flujo y la secuenciación de ARN. También se muestran las colecciones de pelajes de plasma y buffy.

En este protocolo, nuestro objetivo es proporcionar un tutorial paso a paso fácil de seguir sobre cómo recolectar y almacenar células mononucleares y de sangre periférica de alta viabilidad, o PBMC, de sangre completa. Las PBMC incluyen linfocitos, células dendríticas y monocitos. Las PBMC a granel se pueden procesar para aislar tipos de células individuales y se utilizan comúnmente en una amplia variedad de experimentos y ensayos, incluida la secuenciación de ARN y la citometría de flujo.

Además de los PBMC, también demostramos cómo recolectar plasma EDTA y capa leucocitaria completa. Antes de comenzar este protocolo, prepare los materiales y reactivos enumerados en la Tabla 2. Tenga en cuenta que este protocolo está diseñado para la recolección de PBMC de seis tubos de centrifugación en gradiente de densidad, cada uno de los cuales produce aproximadamente 12 millones de células.

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