Biological Evaluation of the MILLIPLEX^® Mouse High Sensitivity T Cell Multiplex Panel

• The Role of Inflammation and Cytokines
• Evaluating a High Sensitivity T Cell Multiplex Mouse Panel
• Evaluation Results
• Powerful Cytokine Profiling with MILLIPLEX^® Mouse Panel
• Materials

The evaluation of cytokine expression is important in research because of the role inflammation plays in many disease states. See how the MILLIPLEX^® Mouse High Sensitivity T Cell Multiplex Panel was evaluated for studying low-level cytokine expression and also quantifying multiple cytokine secretion levels simultaneously in a biologically relevant context.

The Role of Inflammation and Cytokines

Low levels of inflammation are involved in many clinical and sub-clinical disease states such as autoimmune diseases, metabolic diseases, and cancer. Consequently, research investigating low levels of cytokine expression plays a significant role in achieving a deeper understanding of the immune system and its multi-faceted response to most antigens, especially those responses that make up the immune cell-mediated inflammatory processes. Measuring picogram levels of cytokines is critical for understanding their pathogenesis. Model organisms, such as mice, offer unique challenges of sample availability and detection of low levels of cytokines.

Evaluating a High Sensitivity T Cell Multiplex Mouse Panel

A mouse high sensitivity T cell panel multiplex assay was developed for simultaneous measurement of 18 mouse cytokines using Luminex^® xMAP^® technology. Eighteen mouse cytokine levels were analyzed in 25 µL of sample for both in vitro, with PMA-, PHA-, LPS-, Con-A, or calcium ionophore challenged peripheral blood mononuclear cell (PBMC) media samples, and in vivo, using LPS challenged mice, obese mouse models, and an aged-mouse model in serum samples.

Typically, the response of mouse PBMCs to in vitro stimulants is difficult to study due to low levels of cytokine secretion. Using the MILLIPLEX^® Mouse High Sensitivity T Cell Panel, distinctly different stimulant-dependent cytokine responses were observed. The panel also detected low levels of serum cytokines in vivo using LPS-challenged mice (early onset of inflammation), obese mice (OB/OB and DB/DB,) and an aged-mouse model (cytokine profiles of young vs. old mice).

Immunoassay Protocol

The multiplex assay was performed in a 96-well plate. The detailed procedure is as follows:

• Wet the plate with 150 µL wash buffer for 10 minutes and decant.
• Add 50 µL standards or 25 µL samples and 25 µL assay buffer.
• Add 25 µL beads to all wells and incubate overnight at 4°C.
• Wash the beads three times then add 50 µL biotinylated detection antibody cocktail and incubate at room temperature for 1 hour.
• Add 50 µL Streptavidin-Phycoerythrin and further incubate at room temperature for 30 minutes.
• Wash beads three times, add 150 µL sheath fluid, and read on Luminex^® instrumentation (in this case the Luminex^® 200™ system).

Evaluation Results

Standard Curves

Details on the standard curves from the MILLIPLEX^® Mouse High Sensitivity T Cell Panel can be found in Table 1 and Figure 1.

Table 1. Standard curve ranges, sensitivity, precision, accuracy, and linearity for all 18 analytes of the MILLIPLEX^®Mouse High Sensitivity T Cell Panel.

Detection of Analytes in Mouse PBMCs

The high sensitivity panel was able to detect low levels of analytes in mouse PBMC culture media as shown in Table 2.

Table 2 . MILLIPLEX^® Mouse High Sensitivity T Cell Panel detects low levels of analytes in mouse PBMC culture media. PBMCs (Bioreclamation) were thawed, washed, and resuspended in RPMI media (Gibco) containing 10% FCS and 1% Penicillin/ Streptomycin at 10⁶ cells/mL. The PBMCs were aliquoted at 10⁶ cells/well and incubated overnight at 37°C. The next day either PMA, A23187, LPS, Con A, PHA, or all, were added for final concentrations of 0.025, 0.01, 1, 5, or 20 µg/mL, respectively. Conditioned media was collected at various time points, centrifuged at 15,000 RPM for 10 min, and the cell-free supernatants were stored at -80°C prior to assaying in a multiplex format.

Diabetic Mouse Model

Data from using the multiplex panel in a diabetic mouse model is shown in Figure 3.

Aging Mouse Model

Data from using the multiplex panel in an aging mouse model is shown in Figure 4.

For Research Use Only. Not For Use In Diagnostic Procedures.