This protocol describes the quantification of multiple cytokine targets simultaneously in tissue culture supernatants collected from stimulated mouse splenocytes using multiplex bead based immunoassay platform and a flow cytometer.
Bead-based immunoassays employ the same basic principle as sandwich immunoassays. Capture beads, which can be differentiated by size and internal allophycocyanin (APC) fluorescence intensity, are conjugated to antibodies specific to a particular analyte. Next, a selected panel of defined capture bead sets is incubated with a biological sample containing target analytes specific to the capture antibodies. A biotinylated detection antibody cocktail is added, which leads to the formation of capture bead-analyte-detection antibody sandwiches.
Finally, streptavidin-phycoerythrin (SA-PE) is added, which binds to biotinylated detection antibodies, providing fluorescent signal intensities in proportion to the amount of bound analyte. The PE fluorescent signal of analyte-specific beads regions is quantified using flow cytometry, and the concentrations of particular analytes are determined using data analysis software and the standard curve generated in the assay.
In this experiment, we use a mouse T helper cytokine panel to simultaneously quantify the concentration of 13 separate cytokine targets in tissue culture supernatants collected from mouse splenocytes cultured under various stimulatory conditions.
In their role as soluble signaling molecules, cytokines mediate the highly coordinated and multifactorial processes that govern host immunological responses. They are expressed during all stages of the inflammatory process, from initiation to resolution, and regulate a complex interaction network that includes their own synthesis and that of their cellular receptors1. This communication network is layered with a complexity that goes beyond the synergistic or antagonistic relationships that may exist between individual components. Indeed, many cytokines are known to share redundant or at least partially overlapping functions2,3.
Integrated systems biology approaches that simultaneously quantify multiple cytokine analytes are currently providing an increasingly comprehensive understanding of the unique cytokinomes that orchestrate the immune responses underlying multiple disease states4,5. These disease states range from generalized inflammation to cancer, neuro-degenerative conditions, and cardiovascular disease6,7,8,9.
Such cytokine networks can be interrogated effectively using LEGENDplex bead-based immunoassays. These assays are based on the same principle as the sandwich Enzyme-Linked Immunosorbent Assay (ELISA), and use fluorescence encoded microspheres with capture antibodies covalently attached to their surface. These antibodies are immobilized on surface areas much smaller than those required by traditional ELISA formats. This enables such assays to be performed with far less sample volume, while at the same time reducing non-specific binding and providing multiplexed analysis of several analytes.
The assay described in this protocol utilizes this technology to quantitate up to 13 targets simultaneously. Data from this assay can be obtained using a wide variety of commonly available flow cytometers and unlike other available assays does not require the use of dedicated assay-specific instrumentation. With an expanding catalog of validated analyte panels, these assays have been used in several ongoing biomedical research projects10,11,12,13.
To demonstrate the ease and utility of the assay format, in this experiment we use a Mouse T helper cytokine panel to quantitate 13 separate cytokine concentrations from tissue culture supernatants obtained from mouse splenocytes cultured under multiple stimulatory conditions. In addition to tissue culture supernatants, this assay can also be performed using serum or plasma samples.
The assay format described in this protocol can provide robust quantification of soluble mediator profiles in biological samples provided the experimenter has good laboratory technique and adheres to the recommended protocol.
There are several key steps that must be followed in order to guarantee reliable results. First, the recombinant standards must be allowed to reconstitute in assay buffer the full time recommended, and after only diluted in polypropylene tubes. Polystyrene tubes can promote the adherence of the proteins to the wall of the tubes, which can lead to low signals when generating the standard curve. Second, proper shaking during incubation steps is critical. Without proper agitation, the binding characteristics of the assay beads can be severely hindered. In addition, proper washing between incubation steps is also required. The experimenter must ensure that excess reagents are removed from the wells prior to commencing the next step in the protocol. Instruments settings for the flow cytometer used to interrogate the assay beads must be optimized as well. In particular, PMT settings must be adjusted to ensure proper bead separation and broad dynamic ranges for the standard curves. Finally, just prior to being analyzed on the cytometer, beads must be vortexed to insure proper suspension and to avoid the formation of aggregates which can skew results.
This protocol can potentially be modified to analyze tissue homogenates as well as the sample types discussed in this manuscript, provided the researcher is willing to optimize their lysis protocol prior to performing large, full plate assays. The actual technique will of course vary depending on tissue type; however, in general the protocol should use a neutral pH buffer containing physiological concentrations of ionic salts, no denaturing chemicals, and preferably no detergents. If detergent must be used, non-ionic detergent concentrations should be kept at a minimum (e.g. no more than 1%). The buffer should also contain sufficient protease inhibitors to prevent proteolytic degradation of target proteins. Regardless of the lysis protocol used, the final preparations should be centrifuged to remove particulates prior to analysis.
Regarding assay limitations, the concentrations of analyte targets in biological sample types can vary greatly. In order to properly quantify analyte concentrations, they must fall within the top and bottom values for the standard curve. Extrapolation of values of the curve is not necessarily reliable and not recommend. Investigators therefore must optimize dilutions of their particular samples in pilot experiments prior to running a full-plate assay. In addition, the careful preparation of the biological samples to be assayed is paramount to the success of this assay format. Samples that are lipemic, hemolyzed, or contain protein debris will affect the antigen antibody interactions that define the core principle of this immunoassay, and render results which are uninterpretable.
This assay is significant with respect to existing quantification methods for several reasons. When run properly, the assay format can quantitate up to 13 analytes from a sample using far smaller volumes than would be required to analyze the sample using traditional ELISA assays. This assay format also does not require dedicated instrumentation in order to be performed. This is an advantage compared to other commercially available bead-based immunoassays as the assay can be run using any number of commonly used flow cytometers.
Scientific inquiry into the role played by secreted factors and cytokine networks in health and disease is expanding. The assay format described in this manuscript can be a valuable tool for researchers seeking to understand these multifaceted and complex phenomena. Active biomedical research programs are beginning to explore the role of cytokine networks in not only areas such as generalized inflammation6, but also in context of specific disease states such as atherosclerosis, cancer, and neuroinflammation15,16,17. Undoubtedly, investigation will continue to grow in these areas as the search for novel therapeutics to treat these chronic conditions expands. The need for accurate and reliable quantification of the soluble mediators associated with these diseases will remain a critical research priority.
The authors have nothing to disclose.
The authors would like to acknowledge the contributions of the Biomarkers and Immuno-assays Product Development team to the development of the assay. In addition, we would like to thank Vigene Tech, Inc. for their collaborative efforts in designing the data analysis software packages.
Allegra 6R Centrifuge with MICROPLUS Carrier Adaptor | Beckman Coulter | 366816 | For LEGENDplex assays is run in microtubes, V-or U-bottom 96-well plate (optional) |
LEGENDplex Mouse T helper Cytokine Panel | BioLegend | 740005 | Multiplex Immunoassay (13-plex) |
Anti-mouse CD3ε antibody | BioLegend | 100314 | Clone 145-2C11, low endotoxin, azide free format |
Anti-mouse CD28 antibody | BioLegend | 102112 | Clone 37.51, low endotoxin, azide free format |
Vacuum Pump | EMD Millipore | WP6111560 | For LEGENDplex assays using filter plates (recommended) |
Vacuum Manifold | EMD Millipore | MSVMHTS00 | For LEGENDplex assays using filter plates (recommended) |
Sterile Disposable Reagent Reservoirs | Fisher Scientific | 07-200-130 | Or equivalent |
Polypropylene MicroFACS Tubes | Fisher Scientific | 11-842-90 | For LEGENDplex assays is run in microtubes, V-or U-bottom 96-well plate (optional) |
Pipette Kit | Fisher Scientific | 14-388-100 | Four pipette sizes (0.2-2µL, 2-20µL, 20-200µL, 100-1000µL) |
Multi-Channel Pipette | Fisher Scientific | FA10011G | capable of dispensing 5 μL to 200 μL |
Microplate Shaker | Fisher Scientific | 88880023 | Or equivalent |
Microcentrifuge | Fisher Scientific | 75002410 | Or equivalent |
FACS tubes | Fisher Scientific | NC9885747 | 12 x 75 mm round bottom |
PMA (Phorbol 12-myristate 13-acetate) | Sigma-Aldrich | P8139 | |
Lipopolysaccharide (LPS) | Sigma-Aldrich | L-8274 | Escherichia coli O26:B6 |
Ionomycin | Sigma-Aldrich | I0634 | |
Branson B200 Ultrasonic Cleaner | Sigma-Aldrich | Z305359 | Or equivalent |
Microcentrifuge tubes | Sigma-Aldrich | Z666505 | 1.5 mL polypropylene tubes |
Flow Cytometer | Various | Various | Cytometer equipped with single laser (488nm blue) or two lasers (488nm blue or 532nm green + 633nm Red) capable of reading emission wavelengths at 575nm and 660nm |
96-Well Polypropylene Plate | VWR | 82050-662 | For LEGENDplex assays is run in microtubes, V-or U-bottom 96-well plate (optional) |