Elsevier

Methods

Volume 158, 1 April 2019, Pages 22-26
Methods

Method to quantify cytokines and chemokines in mouse brain tissue using Bio-Plex multiplex immunoassays

https://doi.org/10.1016/j.ymeth.2019.02.007Get rights and content

Highlights

  • Multiplex ELISA platforms can measure various protein quantities from a single, small volume of sample. Here, we describe the analysis of 33 different cytokines and chemokines from inflamed brain tissue harvested from mice infected with the parasite Plasmodium berghei – a pathogen that induces a disease referred to as cerebral malaria.

Abstract

This protocol describes how to prepare mouse brain tissue for quantification of multiple inflammatory mediators using a multiplex bead-based immunoassay. It is important to have methods that allow quantification of multiple analytes from small amounts of tissue. Bio-Plex is a Luminex xMAP-based multiplex bead-based immunoassay technology that permits simultaneous analysis of up to 100 analytes from a single tissue sample. This assay has been used extensively to investigate analytes in plasma and serum samples as well as cultured and primary cells. Here, we describe a method for simultaneous analysis of 33 different inflammatory cytokines and chemokines from mouse brain tissue using the Bio-Plex Pro Mouse Chemokine Panel 33-Plex.

Introduction

Since the discovery of the first cytokine, interferon-alpha, more than 60 years ago [1], >300 additional cytokines, chemokines, and growth factors have been discovered and studied extensively [2]. However, the complexities of intracellular and extracellular signaling networks generated by these immune mediators are not entirely understood. It is therefore important to conduct more in-depth research into how these mediators function, especially in combination, under both steady state and inflammatory conditions. Simultaneous analysis of multiple immune mediators offers a more comprehensive understanding of disease processes, immune responses, and therapeutic interventions [3], [4], [5]. This type of analysis is now feasible with commercially available multiplex cytokine and chemokine assays. These assays allow researchers to analyze an entire network of cytokines/chemokines in a single sample, which conserves tissue, streamlines workflows, and accelerates research.

Another advantage of multiplex assays is evident when tissue samples are limited, such as when working with small animal models. Use of a conventional ELISA-based approach to analyze multiple immune mediators one-by-one requires large sample volumes. These large samples are often not available when processing small tissues or specific anatomical regions within a tissue. It is therefore important to have methods that allow simultaneous analysis of immune networks with high reproducibility using small sample volumes. Multiplex immunoassays offer a solution to this problem by surveying dozens of analytes simultaneously. These assays can be used to quantify analytes in plasma/serum samples, cultured/primary cells, tissue samples, cerebrospinal fluid, saliva, and sputum, among others. In addition to saving time and money, multiplex immunoassays help advance vaccine development, drug discovery, basic research, and clinical trials by providing a quantitative snapshot of immune mediators expressed in samples of interest [6].

Multiplex ELISA platforms can measure various protein quantities from a single, small volume of sample. Here, we describe the analysis of 33 different cytokines and chemokines from inflamed brain tissue harvested from mice infected with the parasite Plasmodium berghei – a pathogen that induces a disease referred to as cerebral malaria. While we focus here on the preparation of mouse brain tissue samples, we anticipate that this protocol can be utilized for the preparation of other samples and tissue types.

Proper planning and timing are crucial for the proper execution of this protocol. For optimal results, it is important to plan and allow sufficient time to perform instrument validation/calibration, design plate layouts, and perform mixing/dispensing steps with precision. We cannot overstate the importance of using calibrated pipettors (preferably multichannel) when dispensing the small volumes required for this assay.

Section snippets

Before you begin running the assay

  • 2.1.

    High-level workflow and reagents needs overview

    • 2.1.1.

      Add 50 µl 1× beads to wells

    • 2.1.2.

      Wash buffer: 2 × 100 µl

    • 2.1.3.

      Add 50 µl standards, samples and controls; incubate on shaker at 850 rpm for 30 min

    • 2.1.4.

      Wash buffer: 3 × 100 µl

    • 2.1.5.

      Add 25 µl 1× detection antibody; incubate on shaker at 850 rpm for 30 min

    • 2.1.6.

      Wash buffer: 3 × 100 µl

    • 2.1.7.

      Add 50 µl 1× streptavidin-PE; incubate on shaker at 850 rpm for 10 min

    • 2.1.8.

      Wash buffer: 3 × 100 µl

    • 2.1.9.

      Resuspend in 125 µl assay buffer; shake for 30 s

    • 2.1.10.

      Acquire data on Bio-Plex system.

  • 2.2.

    Plan the plate layout

    • 2.2.1.

      A

Materials and methods

  • 3.1.

    Mouse treatment

    • 3.1.1.

      Adult 8-week-old C57BL/6J (B6) mice used in this study were purchased from the Jackson Laboratories (Bar Harbor, ME). Plasmodium berghei ANKA (PbA) was maintained as previously reported [7]. Animals were infected intraperitoneally with 106 parasitized red blood cells. Parasitemia in each animal was measured by staining 1 µl of blood with Hoechst (1:1000) as previously described [7].

    • 3.1.2.

      To deplete CD8+ T cells, mice were injected intraperitoneally with 500 µg of anti-CD8 depleting

Results

For this study, we infected two cohorts of mice (non-depleted and CD8+ T-cell depleted mice) with Plasmodium berghei and evaluated chemokine and cytokine expression using the Bio-Plex Luminex xMAP-based multiplex bead-based immunoassay. This assay was performed to identify the changes in chemokine and cytokine levels within the brain during experimental cerebral malaria (ECM). We also wanted to determine the role of CD8+ T cells in driving the inflammatory expression profile. Presented here are

Conclusions

Here, we describe a method for preparing mouse brain tissue samples to determine the concentrations of multiple cytokine/chemokine analytes using the Bio-Plex mouse 33-plex panel. For this study, we evaluated three cohorts of mice (naive, PbA-infected and PbA-infected with CD8 T-cell depletion) and evaluated the inflammatory profile within these mice. We observed elevated chemokine and cytokine levels in the brains of infected mice during development of cerebral malaria. Twenty different

Appendices (Materials List)

  • 6.1.

    Instrumentation

    • 6.1.1.

      MP Biomedical FastPrep-24 5G Homogenizer

    • 6.1.2.

      Bio-Rad Bio-Plex 200 System

    • 6.1.3.

      Bio-Plex Pro Wash Station (Bio-Rad Cat. #30034376)

    • 6.1.4.

      Eppendorf Thermomixer C with plate insertion (Cat. #2231000574).

    • 6.1.5.

      Eppendorf Centrifuge 5415 R (Cat. #EP-5415R)

    • 6.1.6.

      Pipet-Lite Multi Pipette L8-200XLS + 20 µl − 200 µl multichannel pipette (Rainin Cat. #17013805)

  • 6.2.

    Reagents and consumables

    • 6.2.1.

      Bio-Plex Sheath Fluid (Bio-Rad Cat #171000055)

    • 6.2.2.

      Bio-Plex Validation Kit 4.0 (Bio-Rad Cat #171203001)

    • 6.2.3.

      Bio-Plex Calibration Kit (Bio-Rad Cat

Additional suggestions

  • 7.1.

    Remember to incubate the reconstituted Standards on ice for 30 min. Time is critical in this step for consistency.

  • 7.2.

    Recall two diluent solutions provided in the assay kit:

    • 7.2.1.

      Standard Diluent. Used for making dilutions with the standards.

    • 7.2.2.

      Sample Diluent. Used for making sample dilutions with serum and plasma samples. The goal here is to match the sample matrix to a first approximation.

  • 7.3.

    For the preparation of the Bio-Plex Quality Control (Section 3.3.1.1), FBS is used here as a carrier

Funding

This work was supported by the Intramural Research Program of the National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), and through collaboration with Bio-Rad Laboratories.

Acknowledgments

No acknowledgements.

References (10)

There are more references available in the full text version of this article.

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