Apr 08, 2026
MSD Proinflammatory cytokine Panel 1 (mouse) Kit
- 1KU Leuven
Protocol Citation: Aisha Sati, Hanne Dhondt 2026. MSD Proinflammatory cytokine Panel 1 (mouse) Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwnqyzvmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 24, 2026
Last Modified: April 08, 2026
Protocol Integer ID: 244114
Keywords: ASAPCRN, msd proinflammatory cytokine panel, plex proinflammatory panel, mouse, plex, kit this protocol, kit instruction
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000458
Abstract
This protocol summarizes the kit instructions to use the V-PLEX Proinflammatory Panel 1 (mouse) Kit.
Guidelines
Introduction:
MSD offers V-PLEX® assays for customers who require unsurpassed performance and quality. V-PLEX products are developed under rigorous design control and are fully validated according to fit-for-purpose principles14 in accordance with MSD’s Quality Management System. They offer exceptional sensitivity, simple protocols, reproducible results, and lot-to-lot consistency. In addition to the analytical validation, the robustness of the assay protocol is assessed during development along with the stability and robustness of the assay components and kits. V-PLEX assays are available in both single-assay and multiplex formats.
The V-PLEX assay menu is organized by panels. Grouping the assays into panels by species, analytical compatibility, clinical range, and expected use ensures optimal and consistent performance from each assay while still providing the benefits and efficiencies of multiplexing. V-PLEX panels are provided in MSD’s MULTI-SPOT® 96-well plate format. The composition of each panel and the location of each assay (i.e., its spot within the well) are maintained from lot to lot. Most individual V-PLEX assays are provided on MSD’s single- spot, 96-well plates. The remaining are provided on the multiplex panel plate.
The Proinflammatory Panel 1 (mouse) measures ten cytokines that are important in inflammation response and immune system regulation as well as numerous other biological processes. These assays can detect secreted biomarkers in a variety of tissues and body fluids where over- or under-expression may indicate a shift in biological equilibrium. This panel also includes assays for many of the Th1/Th2 pathway biomarkers. The Proinflammatory Panel 1 (mouse) measures biomarkers that are associated with a number of disorders, including rheumatoid arthritis,1 Alzheimer’s disease,2 asthma,3 atherosclerosis,4 allergies,5 systemic lupus erythematosus,6 obesity,7 cancer,8 depression,9 multiple sclerosis,10 diabetes,11 psoriasis,12 and Crohn’s disease.13 As a result of their association with such a wide spectrum of disease, these biomarkers are the subjects of drug discovery projects and basic research. The biomarkers constituting the Proinflammatory Panel 1 (mouse) kit are: interferon gamma (IFN-), interleukin-1 beta (IL-1), interleukin-2 (IL-2), interleukin-4 (IL- 4), interleukin-5 (IL-5), interleukin-6 (IL-6), KC/GRO, interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), and tumor necrosis factor alpha (TNF-).
Principle of the Assay:
MESO SCALE DISCOVERY® cytokine assays provide a rapid and convenient method for measuring the levels of protein targets within a single, small- volume sample. The assays in the Proinflammatory Panel 1 (mouse) are sandwich immunoassays. MSD provides a plate pre-coated with capture antibodies on independent and well-defined spots, as shown in the layouts below. Multiplex assays and the individual IL-4, IL-5, KC/GRO, IL-10, and IL-12p70 assays are provided on 10-spot MULTI-SPOT plates (Figure 1); the individual IFN-, IL- 1β, IL-2, IL-6, and TNF-α assays are provided on Small Spot plates (Figure 2). The user adds the sample and a solution containing detection antibodies conjugated with electrochemiluminescent labels (MSD SULFO-TAG™) throughout one or more incubation periods. Analytes in the sample bind to capture antibodies immobilized on the working electrode surface; recruitment of the detection antibodies by the bound analytes completes the sandwich. The user adds an MSD buffer that creates the appropriate chemical environment for electrochemiluminescence (ECL) and loads the plate into an MSD instrument where a voltage applied to the plate electrodes causes the captured labels to emit light. The instrument measures the intensity of emitted light (which is proportional to the amount of analyte present in the sample) and provides a quantitative measure of each analyte in the sample. V-PLEX assay kits have been validated according to the principles outlined in “Fit-for-Purpose Method Development and Validation for Successful Biomarker Measurement” by J. W. Lee, et al.14
1. IFN-
2. IL-1
3. IL-2
4. IL-4
5. IL-5
6. IL-6
7. KC/GRO
8. IL-10
9. IL-12p70
10. TNF-
Figure 1. Multiplex plate spot diagram showing placement of analyte capture antibodies. The numbering convention for the different spots is maintained in the software visualization tools, on the plate packaging, and in the data files.
Figure 2. Small Spot plate diagram showing placement of analyte capture antibodies.
Alternate Protocols:
The suggestions below may be useful as alternate protocols; however, not all were tested using multiple kit lots.
- Alternate Protocol 1, Extended Incubation: Incubating samples overnight at 2-8 ºC may improve sensitivity for some assays. See Appendix A for specific assays that may benefit from this alternate protocol.
- Alternate Protocol 2, Reduced Wash: For tissue culture samples, you may simplify the protocol by eliminating one of the wash steps. After incubating diluted sample, calibrator, or control, add detection antibody solution to the plate without decanting or washing the plate. See Appendix A for assay performance using this protocol.
- Alternate Protocol 3, Dilute-in-Plate: To limit sample handling, you may dilute samples and controls in the plate. For 2-fold dilution, add 25 µL of assay diluent to each sample/control well, and then add 25 µL of neat control or sample. Calibrators should not be diluted in the plate; add 50 µL of each calibrator directly into empty wells. Tests conducted according to this alternate protocol produced results that were similar to the recommended protocol (data not shown).
Appendix A: Calibration curves below illustrate the relative sensitivity of each assay under Alternate Protocols: Reference Protocol (2-hour sample incubation/2 wash steps, blue curve), Alternate Protocol 1 (overnight sample incubation, red curve), and Alternate Protocol 2 (tissue culture: single wash, green curve).
Table 13. Relative sensitivity when using alternate protocols
Assay Characteristics:
Validation
V- PLEX products are validated following fit-for-purpose principles14 and MSD design control procedures. V-PLEX assay components go through an extensive critical reagents program to ensure that the reagents are controlled and well characterized. Before the release of each V-PLEX panel, at least three independent kit lots are produced. Using results from multiple runs (typically greater than 50) and multiple operators, these lots are used to establish production specifications for sensitivity, specificity, accuracy, and precision. During validation, each assay is analytically validated as a singleplex and is also independently evaluated as a multiplex component by running the full multiplex plate using only the single detection antibody for that assay. These results are compared with the results from the multiplex panel when using all detection antibodies. This demonstrates that each assay is specific and independent, allowing them to be multiplexed in any combination. The COA provided with each kit outlines the kit release specifications for sensitivity, specificity, accuracy, and precision.
- Dynamic Range
Calibration curve concentrations for each assay are optimized for a maximum dynamic range while maintaining enough calibration points near the bottom of the curve to ensure a proper fit for accurate quantification of samples that require high sensitivity.
- Sensitivity
The lower limit of detection (LLOD) is a calculated concentration corresponding to the average signal 2.5 standard deviations above the background (zero calibrator). The LLOD is calculated using results from multiple plates for each lot, and the median and range of calculated LLODs for a representative kit lot are reported in this product insert. The upper limit of quantification (ULOQ) and lower limit of quantification (LLOQ) are established for each lot by measuring multiple levels near the expected LLOQ and ULOQ levels. The final LLOQ and ULOQ specifications for the product are established after the assessment of all validation lots.
- Accuracy and Precision
Accuracy and precision are evaluated by measuring calibrators and matrix-based validation samples or controls across multiple runs and multiple lots. For most assays, the results of control measurements fall within 20% of the expected concentration for each run. Precision is reported as the coefficient of variation (CV). Intra-run CVs are typically below 7%, and inter-run CVs are typically below 15%. Rigorous management of inter-lot reagent consistency and calibrator production results in typical inter-lot CVs below 10%. Validation lots are compared using controls and at least 40 samples in various sample matrices. Samples are well correlated with an inter-lot bias typically below 10%.
- Matrix Effects and Samples
Matrix effects from serum, plasma, urine, and cell culture media are measured as part of development and validation. Dilution linearity and spike recovery studies are performed on individual samples rather than pooled samples to assess the variability of results due to matrix effects. The sample dilution suggested in the protocol gives an appropriate dilution factor for all assays in the multiplex. Some assays may benefit from lower or higher dilution factors, depending on the samples and application (data are provided in this product insert). In addition to the matrices listed above, blood, PBMCs, and/or cell lines that have been stimulated to generate elevated levels of analytes are tested. Results confirm that measurement of native proteins at concentrations that are often higher than those found in individual native samples.
- Specificity
The specificity of both capture and detection antibodies is measured during assay development. Antibody specificity is assessed by first running each assay using the multiplex plate with assay-specific detection antibody and assay-specific calibrator. These results are compared to the assay’s performance when the plate is run 1) with the multi-analyte calibrator and assay-specific detection antibodies and 2) with assay-specific calibrator and all detection antibodies. For each validation lot and product release, assay specificity is measured using a multi-analyte calibrator and individual detection antibodies. The calibrator concentration used for specificity testing is chosen to ensure that the specific signal is greater than 50,000 counts.
In addition to measuring the specificity of antibodies to analytes in the multiplex kit, specificity and interference from other related markers are tested during development. This includes the evaluation of selected related proteins and receptors or binding partners.
- Assay Robustness and Stability
The robustness of the assay protocol is assessed by examining the boundaries of the selected incubation times and evaluating the stability of assay components during the experiment and the stability of reconstituted lyophilized components during storage. For example, the stability of reconstituted calibrator is assessed in real-time over a 30-day period. Assay component (calibrator, antibody, control) stability was assessed through freeze–thaw testing and accelerated stability studies. The validation program includes a real-time stability study with scheduled performance evaluations of complete kits for up to 54 months from the date of manufacture.
Representative data from the validation studies are presented in the following sections. The calibration curve and measured limits of detection for each lot can be found in the lot-specific COA that is included with each kit and available for download at www.mesoscale.com.
Analysis of Results:
The calibration curves used to calculate analyte concentrations were established by fitting the signals from the calibrators to a 4- parameter logistic (or sigmoidal dose-response) model with a 1/Y2 weighting. The weighting function provides a better fit of data over a wide dynamic range, particularly at the low end of the calibration curve. Analyte concentrations were determined from the ECL signals by back-fitting to the calibration curve. These assays have a wide dynamic range (4 logs), which allows accurate quantification of samples without the need for multiple dilutions or repeated testing. The calculations to establish calibration curves and determine concentrations were carried out using the MSD DISCOVERY WORKBENCH® analysis software.
Best quantification of unknown samples will be achieved by generating a calibration curve for each plate using a minimum of two replicates at each calibrator level.
Typical Data
Data from the Proinflammatory Panel 1 (mouse) were collected over six months of testing by five operators (63 runs in total). Calibration curve accuracy and precision were assessed for three kit lots. Representative data from one lot are presented below. Data from individual assays are presented in Appendix B. The multiplex panel was tested with individual detection antibodies to demonstrate that the assays are independent of one another. Appendix C compares results for each assay in the kit when the panel is run using the individual detection antibody versus all ten detection antibodies. The calibration curves were comparable. Calibration curves for each lot are presented in the lot-specific COA.
Figure 4. Typical calibration curves for the Proinflammatory Panel 1 (mouse) assay
Appendix B: Single Spot vs Multiplex Plate
The calibration curves below compare assay performance when the assay is run as an individual assay on a single spot plate (blue curve) vs. on the multiplex plate (red curve).
Table 14. Assay performance for individual and 10-plex assays
In general, assays in the single spot format yielded a lower overall signal compared to the 10-plex format. The spots on single-spot plates have a larger binding surface than those on multiplex plates, but the same amount of calibrator was used for each test; therefore, the bound calibrator was spread over a larger surface area reducing the average signal.
Note: Assay performance for IL-4, IL-5, KC/GRO, IL-10, and IL-12p70 are not included since the individual assays are run on multiplex plates.
Appendix C: All vs Single Antibody
The calibration curves below compare results for each assay in the panel when the assays were run on the 10-spot plate using all detection antibodies (blue curve) vs. running each assay using a single, assay-specific detection antibody (red curve).
Table 15. LLODs for detection of a single analyte vs blended antibodies.
As expected, both multiplex formats yielded the same specific signal, but lower background signals were seen when using the single detection antibody.
Sensitivity
- The LLOD is a calculated concentration corresponding to the signal 2.5 standard deviations above the background (zero calibrator). The LLOD shown below was calculated based on 63 runs.
- The ULOQ is the highest concentration at which the CV of the calculated concentration is <25% and the recovery of each analyte is within 75% to 125% of the known value.
- The LLOQ is the lowest concentration at which the CV of the calculated concentration is <25% and the recovery of each analyte is within 75% to 125% of the known value.
- The quantitative range of the assay lies between the LLOQ and ULOQ.
- The LLOQ and ULOQ are verified for each kit lot and the results are provided in the lot-specific COA that is included with each kit and available at www.mesoscale.com.
| A | B | C | D | E | |
| Median LLOD (pg/mL) | LLOD Range (pg/mL) | LLOQ (pg/mL) | ULOQ (pg/mL) | ||
| IFN-γ | 0.04 | 0.01–0.14 | 0.39 | 570 | |
| IL-1β | 0.11 | 0.05–0.23 | 2.04 | 1,030 | |
| IL-2 | 0.22 | 0.07–1.40 | 1.03 | 1,570 | |
| IL-4 | 0.11 | 0.07–0.18 | 0.818 | 1,060 | |
| IL-5 | 0.06 | 0.04–0.07 | 0.302 | 590 | |
| IL-6 | 0.61 | 0.06–1.38 | 7.61 | 3,140 | |
| KC/GRO | 0.24 | 0.13–0.71 | 3.29 | 1,230 | |
| IL-10 | 0.94 | 0.42–2.22 | 7.26 | 2,030 | |
| IL-12p70 | 9.95 | 2.92–25.3 | 179 | 20,600 | |
| TNF-α | 0.13 | 0.05–3.01 | 0.98 | 403 |
Table 5. LLOD, LLOQ, and ULOQ for each analyte in the Proinflammatory Panel 1 (mouse) Kit
Precision
Controls were made by spiking calibrator into mouse serum at three levels within the quantitative range of the assay. Analyte levels were measured by five operators using a minimum of three replicates on 49 runs over five months. Results are shown below. While a typical specification for precision is a concentration CV of less than 25% for controls on both intra- and inter-day runs, for this panel, the data shows most assays are below 15%.
Average intra-run %CV is the average %CV of the control replicates within an individual run. Inter-run %CV is the variability of controls across 49 runs.
Inter-lot %CV is the variability of controls across three kit lots.
| A | B | C | D | E | F | |
| Control | Average Conc. (pg/mL) | Average Intra-run %CV | Inter-run %CV | Inter-lot %CV | ||
| IFN-γ | Control 1 | 740 | 4.4 | 10.9 | 4.7 | |
| Control 2 | 56.1 | 2.2 | 10.3 | 6.6 | ||
| Control 3 | 5.03 | 2.4 | 11.7 | 7.4 | ||
| IL-1β | Control 1 | 1,412 | 2.9 | 8.7 | 2.9 | |
| Control 2 | 105 | 2 | 10.3 | 5.3 | ||
| Control 3 | 9.31 | 2.3 | 12.5 | 7.5 | ||
| IL-2 | Control 1 | 2,504 | 2.5 | 8.4 | 6 | |
| Control 2 | 187 | 2.6 | 11.1 | 9 | ||
| Control 3 | 14.8 | 2.9 | 11.9 | 6.4 | ||
| IL-4 | Control 1 | 701 | 2.5 | 8.2 | 4.8 | |
| Control 2 | 74 | 2.4 | 9.3 | 6.1 | ||
| Control 3 | 10.7 | 3.1 | 12.5 | 9.6 | ||
| IL-5 | Control 1 | 832 | 2.7 | 12.2 | 6.3 | |
| Control 2 | 53.2 | 2.2 | 11.4 | 8.3 | ||
| Control 3 | 2.88 | 3.1 | 20 | 4.9 | ||
| IL-6 | Control 1 | 5,031 | 2.3 | 10.6 | 4.8 | |
| Control 2 | 542 | 2.6 | 10.4 | 1.1 | ||
| Control 3 | 61.5 | 2.6 | 11.1 | 5.3 | ||
| KC/GRO | Control 1 | 1,922 | 2.2 | 10.1 | 1.8 | |
| Control 2 | 237 | 2.1 | 9.9 | 6.8 | ||
| Control 3 | 25.5 | 2.7 | 10.8 | 9.7 | ||
| IL-10 | Control 1 | 2,730 | 4.1 | 8.3 | 6.3 | |
| Control 2 | 619 | 3.9 | 10.8 | 7.1 | ||
| Control 3 | 137 | 4.2 | 11.8 | 6.1 | ||
| IL-12p70 | Control 1 | 32,794 | 2.2 | 10.6 | 3.1 | |
| Control 2 | 4,494 | 1.8 | 12.1 | 5.4 | ||
| Control 3 | 643 | 2.3 | 12.6 | 6.2 | ||
| TNF-α | Control 1 | 380 | 2.8 | 12.6 | 6 | |
| Control 2 | 103 | 2.1 | 8.5 | 3.6 | ||
| Control 3 | 28.5 | 2.5 | 13.6 | 8.7 |
Table 6. Intra-run and Inter-run %CVs for each analyte in the Proinflammatory Panel 1 (mouse) Kit
Dilution Linearity
To assess linearity, normal mouse serum, EDTA plasma, heparin plasma, citrate plasma, and urine from a commercial source as well as cell culture supernatants were spiked with recombinant calibrators and diluted 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, and 64-fold before testing. Percent recovery at each dilution level was normalized to the dilution-adjusted, 2-fold concentration. The average percent recovery shown below is based on samples within the quantitative range of the assay.
| A | B | C | D | E | F | G | H | I | J | K | L | |
| IFN-γ | IL-1β | IL-2 | IL-4 | IL-5 | ||||||||
| Sample Type | Fold Dilution | Average % Recovery | % Recovery Range | Average % Recovery | % Recovery Range | Average % Recovery | % Recovery Range | Average % Recovery | % Recovery Range | Average % Recovery | % Recovery Range | |
| Serum (N=8) | 4 | 107 | 94–115 | 108 | 99–122 | 106 | 98–115 | 144 | 128–154 | 106 | 92–129 | |
| 8 | 99 | 86–107 | 96 | 82–102 | 100 | 88–110 | 156 | 136–169 | 101 | 85–124 | ||
| 16 | 98 | 86–108 | 98 | 88–105 | 94 | 82–110 | 170 | 145–182 | 99 | 84–126 | ||
| 32 | 95 | 82–101 | 99 | 87–109 | 96 | 78–110 | 171 | 139–190 | 94 | 83–116 | ||
| 64 | 98 | 82–109 | 100 | 88–109 | 97 | 83–111 | 184 | 144–204 | 100 | 86–124 | ||
| EDTA Plasma (N=8) | 4 | 101 | 90–112 | 103 | 98–109 | 98 | 92–106 | 123 | 114–129 | 100 | 90–107 | |
| 8 | 100 | 80–115 | 101 | 86–111 | 94 | 84–104 | 134 | 127–149 | 96 | 79–110 | ||
| 16 | 106 | 78–155 | 100 | 85–112 | 90 | 78–105 | 147 | 138–167 | 95 | 80–114 | ||
| 32 | 103 | 79–124 | 100 | 83–115 | 93 | 82–108 | 149 | 133–177 | 99 | 79–120 | ||
| 64 | 114 | 80–167 | 104 | 79–125 | 94 | 79–109 | 160 | 143–196 | 105 | 82–133 | ||
| Heparin Plasma (N=8) | 4 | 105 | 82–122 | 106 | 100–112 | 101 | 94–112 | 134 | 124–139 | 100 | 89–113 | |
| 8 | 98 | 81–123 | 102 | 97–109 | 95 | 90–104 | 147 | 136–152 | 95 | 87–105 | ||
| 16 | 107 | 91–171 | 100 | 93–111 | 92 | 83–107 | 162 | 153–179 | 93 | 85–110 | ||
| 32 | 103 | 86–161 | 100 | 92–111 | 95 | 81–109 | 159 | 129–174 | 93 | 78–121 | ||
| 64 | 106 | 87–147 | 101 | 94–115 | 93 | 78–108 | 174 | 154–194 | 98 | 81–131 | ||
| Citrate Plasma (N=8) | 4 | 99 | 83–109 | 103 | 95–109 | 102 | 94–111 | 120 | 105–131 | 97 | 94–106 | |
| 8 | 91 | 79–100 | 95 | 85–102 | 94 | 88–103 | 125 | 112–137 | 87 | 76–97 | ||
| 16 | 88 | 71–103 | 92 | 83–101 | 90 | 85–99 | 133 | 112–149 | 83 | 70–102 | ||
| 32 | 83 | 71–96 | 89 | 75–104 | 87 | 74–97 | 129 | 105–147 | 83 | 66–102 | ||
| 64 | 86 | 74–101 | 91 | 73–108 | 87 | 74–98 | 134 | 111–155 | 83 | 58–107 | ||
| Urine (N=6) | 4 | 99 | 93–104 | 104 | 93–110 | 105 | 97–115 | 122 | 118–128 | 98 | 84–114 | |
| 8 | 96 | 87–104 | 102 | 93–111 | 103 | 95–119 | 136 | 129–143 | 97 | 88–115 | ||
| 16 | 96 | 89–101 | 99 | 81–107 | 100 | 94–118 | 149 | 145–153 | 91 | 75–106 | ||
| 32 | 94 | 88–100 | 101 | 94–112 | 103 | 95–124 | 151 | 146–157 | 93 | 82–106 | ||
| 64 | 97 | 86–103 | 102 | 86–112 | 103 | 91–122 | 166 | 160–169 | 95 | 81–105 | ||
| Cell Culture Supernatant (N=4) | 4 | 103 | 100–105 | 105 | 101–108 | 96 | 93–100 | 112 | 108–118 | 96 | 94–98 | |
| 8 | 98 | 96–99 | 102 | 99–107 | 92 | 89–95 | 112 | 108–115 | 93 | 92–97 | ||
| 16 | 100 | 96–104 | 102 | 99–108 | 88 | 87–90 | 115 | 112–122 | 92 | 86–96 | ||
| 32 | 95 | 94–97 | 101 | 97–106 | 92 | 91–93 | 112 | 109–115 | 92 | 85–99 | ||
| 64 | 100 | 96–105 | 104 | 101–107 | 93 | 87–96 | 119 | 111–125 | 95 | 89–100 | ||
Table 7. Analyte percent recovery at various dilutions in each sample type
| A | B | C | D | E | F | G | H | I | J | K | L | |
| IL-6 | KC/GRO | IL-10 | IL-12p70 | TNF-α | ||||||||
| Sample Type | Fold Dilution | Average % Recovery | % Recovery Range | Average % Recovery | % Recovery Range | Average % Recovery | % Recovery Range | Average % Recovery | % Recovery Range | Average % Recovery | % Recovery Range | |
| Serum (N=8) | 4 | 98 | 87–109 | 118 | 105–154 | 120 | 105–142 | 98 | 84–112 | 111 | 100–124 | |
| 8 | 90 | 72–102 | 110 | 96–146 | 117 | 102–135 | 87 | 73–100 | 108 | 99–120 | ||
| 16 | 90 | 74–100 | 106 | 95–136 | 119 | 105–136 | 81 | 65–94 | 109 | 97–123 | ||
| 32 | 94 | 73–112 | 111 | 98–145 | 117 | 101–143 | 78 | 62–90 | 117 | 105–142 | ||
| 64 | 98 | 74–117 | 118 | 103–147 | 126 | 107–159 | 81 | 61–93 | 123 | 107–156 | ||
| EDTA Plasma (N=8) | 4 | 97 | 83–95 | 91 | 83–95 | 103 | 99–117 | 93 | 87–101 | 99 | 90–110 | |
| 8 | 91 | 72–93 | 85 | 72–93 | 101 | 90–118 | 85 | 78–94 | 100 | 88–111 | ||
| 16 | 88 | 64–90 | 82 | 64–90 | 102 | 94–123 | 81 | 75–89 | 98 | 84–112 | ||
| 32 | 91 | 68–106 | 87 | 68–106 | 99 | 85–121 | 81 | 68–92 | 102 | 85–124 | ||
| 64 | 95 | 72–112 | 90 | 72–112 | 102 | 88–127 | 86 | 69–102 | 108 | 89–136 | ||
| Heparin Plasma (N=8) | 4 | 98 | 76–111 | 93 | 76–111 | 111 | 103–127 | 87 | 77–95 | 102 | 95–111 | |
| 8 | 96 | 68–115 | 90 | 68–115 | 107 | 94–122 | 77 | 66–89 | 100 | 89–109 | ||
| 16 | 97 | 65–118 | 86 | 65–118 | 109 | 95–124 | 75 | 66–84 | 100 | 88–109 | ||
| 32 | 101 | 65–122 | 88 | 65–122 | 105 | 90–129 | 74 | 62–87 | 105 | 89–118 | ||
| 64 | 104 | 64–125 | 88 | 64–125 | 111 | 92–131 | 78 | 63–90 | 107 | 91–119 | ||
| Citrate Plasma (N=8) | 4 | 91 | 86–99 | 90 | 77–99 | 99 | 86–106 | 96 | 86–105 | 99 | 92–106 | |
| 8 | 83 | 72–97 | 79 | 64–94 | 89 | 75–99 | 84 | 68–96 | 94 | 88–97 | ||
| 16 | 79 | 67–94 | 73 | 59–86 | 87 | 75–101 | 79 | 52–94 | 91 | 85–95 | ||
| 32 | 80 | 65–99 | 71 | 53–91 | 83 | 71–98 | 76 | 46–98 | 92 | 89–97 | ||
| 64 | 82 | 60–102 | 74 | 54–91 | 88 | 76–103 | 79 | 44–103 | 93 | 87–98 | ||
| Urine (N=6) | 4 | 99 | 95–104 | 85 | 78–95 | 103 | 87–114 | 108 | 96–122 | 98 | 91–107 | |
| 8 | 94 | 83–106 | 70 | 59–79 | 98 | 84–109 | 107 | 87–133 | 96 | 84–112 | ||
| 16 | 93 | 81–101 | 64 | 53–75 | 94 | 74–111 | 106 | 90–132 | 97 | 85–116 | ||
| 32 | 96 | 85–105 | 63 | 49–77 | 91 | 82–106 | 106 | 91–140 | 102 | 89–122 | ||
| 64 | 101 | 92–108 | 64 | 50–78 | 95 | 85–108 | 113 | 101–148 | 107 | 94–130 | ||
| Cell Culture Supernatant (N=4) | 4 | 93 | 91–94 | 76 | 72–80 | 93 | 87–100 | 94 | 92–98 | 93 | 92–94 | |
| 8 | 90 | 88–92 | 69 | 63–72 | 84 | 79–92 | 87 | 85–88 | 89 | 87–93 | ||
| 16 | 88 | 83–91 | 65 | 60–69 | 84 | 77–94 | 83 | 79–88 | 89 | 84–93 | ||
| 32 | 93 | 88–97 | 67 | 62–72 | 81 | 74–90 | 81 | 78–87 | 93 | 88–96 | ||
| 64 | 96 | 90–102 | 70 | 66–76 | 85 | 77–101 | 85 | 79–93 | 96 | 92–100 | ||
Table 7 continued
Note: For the IL-4 assay, dilution linearity performance may be improved by normalizing each dilution point to the 4-fold dilution, rather than to the 2-fold dilution.
Spike Recovery
Spike recovery measurements of different sample types throughout the quantitative range of the assays were evaluated. Multiple individual mouse samples (serum, EDTA plasma, heparin plasma, citrate plasma, and urine) were obtained from a commercial source. These samples, along with cell culture supernatants, were spiked with calibrators at three levels (high, mid, and low) then diluted 2- fold. The average % recovery for each sample type is reported along with %CV and % recovery range.
| A | B | C | D | E | F | G | H | I | J | |
| Citrate Plasma (N=8) | Heparin Plasma (N=7) | EDTA Plasma (N=7) | ||||||||
| Average % Recovery | %CV | % Recovery Range | Average % Recovery | %CV | % Recovery Range | Average % Recovery | %CV | % Recovery Range | ||
| IFN-γ | 109 | 9.8 | 92-131 | 99 | 11.8 | 69-114 | 103 | 18.8 | 84-143 | |
| TNF-β | 102 | 10.8 | 85–123 | 99 | 8.1 | 87–117 | 105 | 11.9 | 90–135 | |
| IL-2 | 99 | 6.2 | 90–111 | 97 | 4.9 | 90–104 | 106 | 4 | 99–117 | |
| IL-4 | 66 | 6.7 | 55–75 | 58 | 6.4 | 48–67 | 67 | 7.9 | 58–81 | |
| IL-5 | 101 | 9.1 | 81–115 | 99 | 10.6 | 79–116 | 102 | 10.9 | 83–126 | |
| IL-6 | 99 | 10.6 | 78–115 | 97 | 11.7 | 80–116 | 105 | 17.1 | 84–143 | |
| KC/GRO | 108 | 9.4 | 91–127 | 100 | 10.3 | 89–129 | 98 | 8.6 | 82–113 | |
| IL-10 | 90 | 6.2 | 77–100 | 92 | 8.8 | 73–111 | 101 | 8.9 | 88–130 | |
| IL-12p70 | 121 | 20.5 | 85–193 | 117 | 13.2 | 91–143 | 131 | 17.1 | 111–194 | |
| TNF-α | 95 | 4.6 | 87–106 | 91 | 5 | 82–99 | 95 | 6.4 | 88–113 | |
Table 8. Spike recovery measurements of different sample types in the Proinflammatory Panel 1 (mouse) Kit
| A | B | C | D | E | F | G | H | I | J | |
| Serum (N=8) | Urine (N=5) | Cell Culture Supernatants (N=4) | ||||||||
| Average % Recovery | %CV | % Recovery Range | Average % Recovery | %CV | % Recovery Range | Average % Recovery | %CV | % Recovery Range | ||
| IFN-γ | 115 | 11.3 | 91–137 | 104 | 8.9 | 82–105 | 108 | 5.2 | 96–112 | |
| IL-1β | 105 | 10.3 | 81–121 | 91 | 6.8 | 80–101 | 103 | 4.6 | 93–108 | |
| IL-2 | 106 | 6.4 | 94–117 | 91 | 8.4 | 85–119 | 106 | 3.5 | 98–111 | |
| IL-4 | 71 | 12.4 | 53–86 | 60 | 12 | 46–71 | 101 | 5.1 | 91–105 | |
| IL-5 | 108 | 7.7 | 92–122 | 96 | 10 | 68–106 | 105 | 6.9 | 93–117 | |
| IL-6 | 107 | 15.3 | 72–131 | 88 | 11.7 | 116–164 | 109 | 2.9 | 103–114 | |
| KC/GRO | 84 | 6.2 | 74–91 | 135 | 9.7 | 84–106 | 128 | 6.6 | 122–141 | |
| IL-10 | 88 | 7.8 | 73–99 | 96 | 6.3 | 79–124 | 121 | 7.5 | 100–128 | |
| IL-12p70 | 124 | 7.7 | 107–144 | 94 | 12.6 | 63–95 | 117 | 4.5 | 112–125 | |
| TNF-α | 91 | 6.9 | 75–101 | 78 | 11.6 | 82–105 | 114 | 4.1 | 105–121 | |
Specificity
To assess specificity, each assay in the panel was tested individually. Nonspecific binding was also evaluated with additional recombinant mouse analytes (IL-13, IL-17, GM-CSF, MCP-1, MIP-3, RANTES, TNF-RI, TNF-RII, and VEGF). Nonspecific binding was less than 0.5% for all assays in the kit.
To evaluate the TNF- assay for interference by its receptors, the Proinflammatory Panel 1 (mouse) was run with TNF--containing serum to which 10 ng/mL of TNF-RI or 15 ng/mL of TNF-RII was added. Assay interference was not observed.
Stability
The reconstituted calibrator, reconstituted controls, and diluents were tested for freeze–thaw stability. Results (not shown) demonstrated that reconstituted calibrator, reconstituted controls, Diluent 41, and Diluent 45 can go through five freeze–thaw cycles without significantly affecting the performance of the assay. Once reconstituted, the multi-analyte calibrator is stable for 30 days at 2–8 °C. Partially used MSD plates may be sealed and stored up to 30 days at 2–8 °C in the original foil pouch with desiccant. Results from control measurements changed by 30% after partially used plates were stored for 30 days. The validation study includes a real-time stability study with scheduled performance evaluations of complete kits for up to 54 months from the date of manufacture.
Calibration
All the assays in the panel are calibrated against a reference calibrator generated at MSD.
MSD reference calibrators for the following analytes were evaluated against the NIBSC/WHO International Standards; the ratios of International Units of biological activity per mL (IU/mL) of NIBSC standard relative to pg/mL of MSD calibrator are shown in the table below. To convert MSD concentrations to biological activity relative to the WHO International Standard, multiply the MSD concentration by the ratio provided.
| A | B | C | |
| Analyte | NIBSC/WHO Catalog Number | NIBSC (IU/mL): MSD (pg/mL) | |
| IL-1β | 96/668 | 1.18 | |
| IL-2 | 93/566 | 1 | |
| IL-4 | 91/656 | 0.89 | |
| IL-6 | 93/730 | 1 | |
| TNF-α | 88/532 | 1 |
Tested Samples:
Normal Samples
Normal mouse serum, EDTA plasma, heparin plasma, citrate plasma, and urine samples from a commercial source were diluted 2-fold and tested. Results for each sample set are displayed below. Concentrations are corrected for sample dilution. Median and range are calculated from samples with concentrations at or above the LLOD.
| A | B | C | D | E | F | G | H | I | J | K | L | |
| Sample Type | Statistic | IFN-γ | IL-1β | IL-2 | IL-4 | IL-5 | IL-6 | KC/GRO | IL-10 | IL-12p70 | TNF-α | |
| Serum (N=16) | Median (pg/mL) | 0.95 | 2.27 | 1.02 | 0.43 | 2.72 | 21.6 | 48.3 | 11 | 81 | 12 | |
| Range (pg/mL) | 0.34–28.7 | 1.13–3.95 | 0.55–3.98 | 0.23–1.10 | 0.58–6.52 | 5.28–111 | 28.7–102 | 5.71–45.4 | 64.8–97.1 | 8.23–34.4 | ||
| % Detected | 100 | 100 | 100 | 94 | 100 | 100 | 100 | 100 | 13 | 100 | ||
| EDTA Plasma (N=15) | Median (pg/mL) | 41.2 | 0.86 | 3.86 | 0.63 | 2.59 | 117 | 70.5 | 56.5 | 69.3 | 38.5 | |
| Range (pg/mL) | 18.6–262 | 0.46–2.40 | 2.60– 5.89 | 0.48–0.70 | 1.50–2.88 | 11.0–185 | 54.2–96.9 | 31.5–74.7 | 50.2–171 | 21.3–47.0 | ||
| % Detected | 100 | 87 | 100 | 60 | 100 | 100 | 100 | 100 | 73 | 100 | ||
| Heparin Plasma (N=15) | Median (pg/mL) | 262 | 1.62 | 4.63 | 0.75 | 4.01 | 175 | 269 | 76.4 | 85.6 | 65.3 | |
| Range (pg/mL) | 156–352 | 0.61–2.25 | 3.35–7.36 | 0.42–1.49 | 2.26–5.72 | 28.8–355 | 220–369 | 63.7–105 | 38.0–152 | 35.0–76.7 | ||
| % Detected | 100 | 87 | 100 | 60 | 100 | 100 | 100 | 100 | 53 | 100 | ||
| Citrate Plasma (N=16) | Median (pg/mL) | 7.04 | 1.01 | 3.09 | 0.73 | 3.37 | 41.9 | 65.3 | 30.7 | 71.2 | 42.8 | |
| Range (pg/mL) | 0.31–122 | 0.45–2.02 | 0.65–5.03 | 0.39–1.47 | 1.72–8.24 | 6.84–74.2 | 34.9–172 | 5.30–68.2 | 50.4–107 | 5.45–58.8 | ||
| % Detected | 100 | 100 | 94 | 100 | 100 | 100 | 100 | 100 | 94 | 100 | ||
| Urine (N=10) | Median (pg/mL) | 0.32 | 0.57 | 0.5 | 0.95 | ND | ND | 2.31 | 1.36 | 102 | 0.63 | |
| Range (pg/mL) | 0.09–0.66 | 0.35–1.34 | 0.49–0.65 | 0.33–1.31 | ND | ND | 1.91–2.84 | 0.98–1.53 | 67.3–125 | 0.48–3.90 | ||
| % Detected | 70 | 60 | 30 | 90 | 0 | 0 | 100 | 40 | 90 | 80 |
Table 10. Normal mouse samples tested in the Proinflammatory Panel 1 (mouse) Kit
ND = Non-detectable
% Detected = % of samples with concentrations at or above the LLOD
Stimulated Samples
Freshly collected, normal, pooled, mouse whole blood was incubated at 37ºC for different time periods either with lipopolysaccharide (LPS) or with peptidoglycan (PG) and zymosan (ZY) as shown below; plasma was isolated at the end of incubations. The dilution-adjusted concentrations (pg/mL) for each stimulation model are displayed below. Assays that showed a significant difference in analyte level with prolonged stimulation are identified with an asterisk.
Figure 6. Normal mouse whole blood stimulated with LPS
Figure 7. Normal mouse whole blood stimulated with PG/ZY
A mouse monocyte macrophage cell line (J774A.1) was stimulated for 4 hours with LPS or pokeweed mitogen (PWM). The lysate was collected and tested. The concentrations were normalized for 50 µg of lysate per well. Analyte levels for IFN-, IL-4, IL-5, and IL-12p70 were non-detectable. Measurements that were above saturation levels are identified with an arrow.
Figure 8. J774A.1 mouse macrophage cell line stimulated with LPS or PWM
Assay Components:
- Calibrators
The assay calibrator blend uses the following recombinant mouse proteins:
| A | B | |
| Calibrator | Expression System | |
| IFN-γ | E. coli | |
| IL-1β | E. coli | |
| IL-2 | E. coli | |
| IL-4 | E. coli | |
| IL-5 | Insect cell line | |
| IL-6 | E. coli | |
| KC/GRO | E. coli | |
| IL-10 | E. coli | |
| IL-12p70 | Insect cell line | |
| TNF-α | E. coli |
Table 11. Recombinant mouse proteins used in the Calibrators
- Antibodies
| A | B | C | D | |
| Source Species | ||||
| Analyte | MSD Capture Antibody | MSD Detection Antibody | Assay Generation | |
| IFN-γ | Rat Monoclonal | Rat Monoclonal | A | |
| IL-1β | Mouse Monoclonal | Goat Polyclonal | A | |
| IL-2 | Rat Monoclonal | Rat Monoclonal | A | |
| IL-4 | Rat Monoclonal | Rat Monoclonal | A | |
| IL-5 | Rat Monoclonal | Rat Monoclonal | A | |
| IL-6 | Rat Monoclonal | Goat Polyclonal | B | |
| KC/GRO | Rat Monoclonal | Goat Polyclonal | A | |
| IL-10 | Rat Monoclonal | Goat Polyclonal | A | |
| IL-12p70 | Rat Monoclonal | Rat Monoclonal | A | |
| TNF-α | Hamster Monoclonal | Goat Polyclonal | B | |
Table 12. Antibody source species
Plate Layout:
| A | B | C | D | E | F | G | H | I | J | K | L | M | |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||
| A | CAL-01 | Sample-01 | Sample-09 | Sample-17 | Sample-25 | Sample-33 | |||||||
| B | CAL-02 | Sample-02 | Sample-10 | Sample-18 | Sample-26 | Sample-34 | |||||||
| C | CAL-03 | Sample-03 | Sample-11 | Sample-19 | Sample-27 | Sample-35 | |||||||
| E | CAL-05 | Sample-05 | Sample-13 | Sample-21 | Sample-29 | Sample-37 | |||||||
| F | CAL-06 | Sample-06 | Sample-14 | Sample-22 | Sample-30 | Sample-38 | |||||||
| G | CAL-07 | Sample-07 | Sample-15 | Sample-23 | Sample-31 | Sample-39 | |||||||
| H | CAL-08 | Sample-08 | Sample-16 | Sample-24 | Sample-32 | Sample-40 | |||||||
| A | B | C | D | E | F | G | H | I | J | K | L | M | |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | ||
| A | CAL-01 | Control 1 | Sample-06 | Sample-14 | Sample-22 | Sample-30 | |||||||
| B | CAL-02 | Control 2 | Sample-07 | Sample-15 | Sample-23 | Sample-31 | |||||||
| C | CAL-03 | Control 3 | Sample-08 | Sample-16 | Sample-24 | Sample-32 | |||||||
| D | CAL-04 | Sample-01 | Sample-09 | Sample-17 | Sample-25 | Sample-33 | |||||||
| E | CAL-05 | Sample-02 | Sample-10 | Sample-18 | Sample-26 | Sample-34 | |||||||
| F | CAL-06 | Sample-03 | Sample-11 | Sample-19 | Sample-27 | Sample-35 | |||||||
| G | CAL-07 | Sample-04 | Sample-12 | Sample-20 | Sample-28 | Sample-36 | |||||||
| H | CAL-08 | Sample-05 | Sample-13 | Sample-21 | Sample-29 | Sample-37 | |||||||
Figure 9. Sample plate layout that can be used for the assays. Each sample, calibrator, and control (Plus Kit) is measured in duplicate in side-by-side wells.
Plate Diagram:
Figure 10. Plate diagram.
Materials
Reagents Supplied With All Kits
| A | B | C | D | E | F | G | H | |
| Reagent | Storage | Catalog No. | Size | Quantity Supplied | Description | |||
| 1-Plate Kit | ||||||||
| Proinflammatory Panel 1 (mouse) Calibrator Blend | 2–8 °C | C0048-2 | 1 vial | 1 vial | Ten recombinant mouse proteins in diluent, buffered and lyophilized. Individual analyte concentration is provided in the lot-specific certificate of analysis (COA). | |||
| Diluent 41 | ≤ -10 °C | R50AH-1 | 10 mL | 1 bottle | Diluent for samples and calibrator. | |||
| R50AH-2 | 50 mL | - | ||||||
| Diluent 45 | ≤ -10 °C | R50AI-1 | 5 mL | 1 bottle | Diluent for detection antibody. | |||
| R50AI-2 | 25 mL | - | ||||||
| MSD GOLD™ Read Buffer B | RT | R60AM-1 | 18 mL | 1 bottle | Buffer to catalyze the electro-chemiluminescence reaction on QuickPlex Ultra™ plates. | |||
| R60AM-2 | 90 mL | - | ||||||
| Read Buffer T (4X) | RT | R92TC-3 | 50 mL | 1 bottle | Buffer to catalyze the electro-chemiluminescence reaction on SECTOR™ plates. | |||
Additional Materials and Equipment:
- Appropriately sized tubes for reagent preparation
- Polypropylene microcentrifuge tubes for preparing dilutions
- Liquid handling equipment for the desired throughput, capable of dispensing 10 to 150 µL/well into a 96-well microtiter plate
- Plate washing equipment: automated plate washer or multichannel pipette
- Microtiter plate shaker (rotary) capable of shaking at 500-1,000 rpm
- Phosphate-buffered saline (PBS) plus 0.05% Tween-20 for plate washing or MSD Wash Buffer catalog no. R61AA-1 (included in V-PLEX Plus kit)
- Adhesive plate seals (3 per plate included in V-PLEX Plus kits)
- Deionized water
- Vortex mixer
- MSD provides 100ml wash Buffer as a 20X stock solution in the V-PLEX Plus kit. Dilute the stock solution to 1X before use.
- MSD provides Read Buffer T as a 4X stock solution. The working solution is 2X. For one plate, combine:
Before start
Best Practices:
- Do not mix or substitute reagents from different sources or different kit lots. Lot information is provided in the lot-specific COA.
- Assay incubation steps should be performed between 20–26 °C to achieve the most consistent signals between runs.
- Bring frozen diluent to room temperature in a 24 °C water bath. Thaw other reagents on wet ice and use as directed without delay.
- Prepare calibrators, samples, and controls in polypropylene microcentrifuge tubes; use a fresh pipette tip for each dilution; vortex after each dilution before proceeding.
- Do not touch the pipette tip on the bottom of the wells when pipetting into the MSD plate.
- Avoid prolonged exposure of detection antibody (stock or diluted) to light. During the antibody incubation step, plates do not need to be shielded from light except for direct sunlight.
- Avoid bubbles in wells at all pipetting steps. Bubbles may lead to variable results; bubbles introduced when adding read buffer may interfere with signal detection.
- Use reverse pipetting when necessary to avoid introduction of bubbles. For empty wells, pipette to the bottom corner.
- Plate shaking should be vigorous with a rotary motion between 500 and 1,000 rpm. Binding reactions may reach equilibrium sooner if you use shaking at the middle of this range (~700 rpm) or above.
- When using an automated plate washer, rotate the plate 180 degrees between wash steps to improve assay precision.
- Gently tap the plate on a paper towel to remove residual fluid after washing.
- If an incubation step needs to be extended, avoid letting the plate dry out by keeping sample or detection antibody solution in the plate.
- Remove plate seals prior to reading the plate.
- Make sure that read buffer is at room temperature when added to a plate.
- Do not shake the plate after adding read buffer.
- To improve inter-plate precision, keep time intervals consistent between adding read buffer and reading the plate. Unless otherwise directed, read the plate as soon as possible after adding read buffer.
- If assay results are above the top of the calibration curve, dilute the samples and repeat the assay.
- When running a partial plate, seal the unused sectors to avoid contaminating unused wells. Remove all seals before reading. Partially used plates may be sealed and stored up to 30 days at 2–8 °C in the original foil pouch with desiccant. You may adjust volumes proportionally when preparing reagents.
Reagent Preparation:
- Bring all reagents to room temperature.
Note
Important: Upon the first thaw, aliquot Diluent 41 and Diluent 45 into suitable volumes before refreezing.
Prepare Calibrator Dilutions
30m
Follow kit instruction to prepare 7 calibrator solutions plus a zero calibrator for up to 4 replicates:
-Prepare the highest calibrator (Calibrator 1) by adding 1000 µL of Diluent 41 to the lyophilized calibrator vial.
-After reconstituting, invert at least 3 times (do not vortex).
-Let the reconstituted solution equilibrate at Room temperature for 00:15:00 - 00:30:00
-Vortex briefly using short pulses.
Prepare the next calibrator by transferring 100 µL of the highest calibrator to 300 µL of Diluent 41.
-Mix well by vortexing.
-Repeat 4-fold serial dilutions 5 additional times to generate 7 calibrators.
-Use Diluent 41 as the zero calibrator.
Dilute Samples
Dilute samples with Diluent 41. For mouse CSF use 2-fold dilution. For primary microglia culture supernatants, dilute 5-fold using Diluent 41.
Prepare Detection Antibody Solution
MSD provides each detection antibody separately as a 50X stock solution. The working solution is 1X. Prepare the detection antibody solution immediately before use as per kit instructions:
For one plate, combine the following detection antibodies and add to 2400 µL of Diluent 45:
- 60 µL of SULFO-TAG Anti-ms IFN- Antibody
- 60 µL of SULFO-TAG Anti-ms IL-1 Antibody
- 60 µL of SULFO-TAG Anti-ms IL-2 Antibody
- 60 µL of SULFO-TAG Anti-ms IL-4 Antibody
- 60 µL of SULFO-TAG Anti-ms IL-5 Antibody
- 60 µL of SULFO-TAG Anti-ms IL-6 Antibody
- 60 µL of SULFO-TAG Anti-ms KC/GRO Antibody
- 60 µL of SULFO-TAG Anti-ms IL-10 Antibody
- 60 µL of SULFO-TAG Anti-ms IL-12p70 Antibody
- 60 µL of SULFO-TAG Anti-ms TNF- Antibody
Assay Protocol
4h
STEP 1: Wash and Add Sample
Wash the plate 3 times with at least 150 µL /well of Wash Buffer.
Add 50 µL of prepared samples, and calibrators per well. Seal the plate with an adhesive plate seal and incubate at Room temperature with shaking for 02:00:00 .
2h
STEP 2: Wash and Add Detection Antibody Solution
Wash the plate 3 times with at least 150 µL /well of Wash Buffer.
Add 25 µL of detection antibody solution to each well. Seal the plate with an adhesive plate seal and incubate at Room temperature with shaking for 02:00:00 .
2h
STEP 3: Wash and Read
Wash the plate 3 times with at least 150 µL /well of Wash Buffer.
Add 150 µL of Read Buffer to each well. Analyze the plate on an MSD instrument. Incubation in Read Buffer is not required before reading the plate.
Plate reading
Read plate on electronic plate reader (MSD Meso QuickPlex SQ 120MM) using methodical mind software.
Protocol references
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12. Yehuda H, et al. Isothiocyanates inhibit psoriasis-related proinflammatory factors in human skin. Inflamm Res. 2012;61:735-42.
13. Gologan S, et al. Inflammatory gene expression profiles in Crohn’s disease and ulcerative colitis: A comparative analysis using a reverse transcriptase multiplex ligation-dependent probe amplification protocol. J Crohns. Epub Sept 26, 2012. (j.crohns.2012.08.015).
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16. Removal of blood from laboratory animals and birds: First report of the BVA/FRAME/RSPCA/UFAW joint working group on refinement. Lab Anim. 1993;27:1-22
Acknowledgements