The Human Immune Monitoring Center (HIMC)

Immunoassays

We have standardized immunoassays for human and mouse cytokines, for analysis of serum, plasma, cell culture supernatants, tissue homogenates and other fluids. We measure multiple cytokines simultaneously using Luminex Bead Array or Olink Arrays which utilizes PEA Technology. Biomarker information in our standard Luminex kits can be downloaded here. For questions about the choice of Luminex vs. Olink, see our FAQ’s here.

Standard Assays (per-sample pricing, no minimum sample number): For human cytokines, we offer a 80-plex Luminex assay custom combination from EMD Millipore panels. We have compared this assay to our legacy 51-plex, for which we have extensive performance data.  For detection of mouse cytokines, we offer a 48-plex Luminex Procarta array by Thermo Fisher Scientific.  Biomarkers in these arrays are displayed below.  

Comment: We maintain inventory for the Human H80, Mouse 48, Human 48 plex. These assays are charged per sample (2 replicates) and include materials and tech labor.

For custom assays we charge: 1. For the “Materials” (full price for kit of choice) and, 2. Tech labor. Custom kit order is placed once we receive a service request in iLab. For more information on custom orders and kit options please contact Holden Maecker: Immunoassay Manager @ maecker@stanford.edu.

Kit Options:

Human 48 plex

CVD-1

CVD-2

CVD-3

CVD-4

Human immunoglobulin Isotyping Panel

Human Metabolic Hormone Panel:

Human Soluble Receptor 14 Plex

Human Immuno-Oncology Checkpoint Panel-1

Rat 27 plex cytokines

NHP

TGF-beta Multi species

For more options, please email maecker@stanford.edu

User Instructions

1.  Planning your assay:  We suggest analyzing all samples in a single experiment, to minimize run to run variability.  To account for plate-to-plate differences and downstream analysis, it is recommended to evenly distribute groups across multiple plates (i.e., don't place all controls on one plate and all test samples on another). However, all time points per subject should be in the same plate.  Samples will be run in the order provided, unless instructed otherwise.  Please consult with us for optimized design and sample distribution in the planning phase of your experiments.

2.  Collecting your samples:  For Human 80 plex we request a minimum of 200 microliters per sample to run duplicate wells without dilution.  Be sure samples are processed and spun before freezing, to remove cells and debris that can clog the Luminex instrument; see our SERUM PROCESSING or PLASMA PROCESSING protocols for details on serum or plasma collection and processing.  Process serum and plasma within two hours of blood draw if possible.  Avoid hemolysis, which interferes with cytokine detection.  Serum and plasma processing services are also available through https://med.stanford.edu/ctru.html. For mouse and rodent samples standard processing vials are available: Kent Scientific &Mini Collect  , we request 50-70ul of plasma or serum samples . For Tissue homogenates please include a buffer with protease inhibitors,  and normalize samples to total protein. For cell supernatants include media with serum as background. If questions, please email: maecker@stanford.edu.  

All samples should be frozen at -80C and transferred to the HIMC on dry ice.  Multiple freeze-thaw cycles should be avoided.  Please label all tubes clearly on the side and top, email an excel list of your samples to maecker@stanford.edu (or post to iLab or Basecamp for existing projects).

3. Running samples:  An online order is required before samples can be run. Turnaround time varies by the project size (number of samples) and the current schedule; Projects with fewer than 20 samples can be completed within two weeks of order and sample delivery. We are located at: 1651 Page Mill Rd. room 0280, 650-723-4984.

Plasma and Serum samples are run with a 1:3 dilution which is optimal for these matrices and prevents clogging of the instrument; dilution of other sample types is optional; it can improve quantitation of high-level cytokines, with minimal impact on the detection of most low-level cytokines.  Leftovers will be discarded unless you provide instructions and will not be saved for more than two weeks.

4.  Data analysis:  Data is shared via our Basecamp project management system.  New customers will receive an email invitation to join the system. An Excel report will be posted there, containing summaries of median fluorescence intensity (MFI), calculated concentration, bead count, and coefficient of variation (CV) of replicate wells, for each cytokine in each sample, standard, and control.  QC notes are also provided in the report.  The data is simultaneously uploaded to our online database, Stanford Data Miner, where it can be integrated with other HIMC data from your lab, as well as clinical/demographic data if provided.  Click the help link in the upper left corner of the Data Miner homepage for instructions on using this system.

In general for Luminex assays, the HIMC recommends analyzing raw data (median fluorescence intensity) rather than concentration (pg/mL). Reasons for this recommendation are many and complex. Most prominent is that standard curves are generated in a matrix that differs from biological matrix of your specimens so, in this sense, standard curves can be biased. Also, standard curves become non-linear at high and low ends, yielding unreliable and biased concentrations.

Normalization (to controls or to global sample median for each plate) is recommended when comparing values across plates.

Description for Publication

Protocols

See the Protocols page for complete HIMC protocols as well as short descriptions suitable for publications and grant applications.

Statistical consultation is available through the HIMC Statistical Consulting Service.

Additional information:

• Methods for collection mouse blood
• BD Vacutainer guide
• Supernatant and Homogenates