Data Analysis

Statistical consultation is available through the HIMC Statistical Consulting Service.

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 arrays or Olink Arrays which utilizes PEA Technology-(https://www.olink.com/products/). Download background information on the protein analytes in our standard kits 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 mouse cytokines, we offer a 48-plex Luminex Procarta array by Thermo Fisher Scientific.  Analytes in these arrays are displayed below:
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H80-Panel 1 (HCYTA-60K-PX48)

H80 Panel-2 ( HCP2MAG-62K-PX23)

EGF

IL-27

6Ckine/CCL21/Exodus-2

IL-28A/IFNλ2

Eotaxin-1/CCL11

IL-3

BCA-1/CXCL13

IL-33/NF-HEV (mature)

FGF-2

IL-4

CTACK/CCL27

LIF

Flt-3 ligand

IL-5

ENA-78/CXCL5

MCP-2/CCL8

Fractalkine/CX3CL1

IL-6

Eotaxin-2 /CCL24/MPIF-2

MCP-4/CCL13

G-CSF

IL-7

Eotaxin-3/CCL26

MIP-1δ/MIP-5/CCL15

GM-CSF

IL-8/CXCL8

I-309/CCL1

SCF

GRO-alpha

IL-9

IL-16

SDF-1/CXCL12

IFN-gamma

IP-10/CXCL10

IL-20

TARC/CCL17

IFN-α2

M-CSF

IL-21

TPO

IL-10

MCP-1/CCL2

 IL-23

TRAIL/TNFSF10

IL-12 (p40)

MCP-3/CCL7

 

TSLP

IL-12 (p70)

MDC (CCL22)

   

IL-13

MIG/CXCL9

H80 Panel-3

 

IL-15

MIP-1α/CCL3

sFAS / TNFRSF6

sICAM-1

IL-17A/CTLA-8

MIP-1β/CCL4

sFasL

sVCAM-1

IL-17E/IL-25

PDGF-AA

MIF

Leptin

IL-17F

PDGF-AB/BB

PAI-1 (total)/Serpine-E

HGF

IL-18

RANTES/CCL5

 

Resistin

IL-1RA

sCD40L

   

IL-1α

TGFα

   

IL-1β

TNF-α

   

IL-2

TNF-β

   

IL-22

VEGF

   

Mouse 48 Plex (Procarta)

BAFF

 IFN-g

 IL-1a

 IL-2Ra

 IL-7

 M-CSF

 BTC

 IL-10

 IL1-b

 IL-3

 IL-7Ra

MIP-1a

 ENA-78

 IL-12p70

IL-2

 IL-31

 IL-9

 MIP-1b

 Eotaxin

 IL-13

 IL-22

 IL-33

 IP-10

 MIP-2

 G-CSF

 IL-15

 IL-23

 IL-33R

 Leptin

 RANKL

 GM-CSF

 IL-17A

 IL-25

 IL-4

 LIF

 RANTES

 GRO-a

 IL-18

 IL-27

 IL-5

 MCP-1

 TNF-a

 IFN-a

 IL-19

 IL-28

 IL-6

 MCP-3

 VEGF-A

Olink Immunoassays:

Olink assays use proximity extension to create a PCR product, read out on the Fluidigm BioMark platform.  There are multiple panels available, each with 92 analytes, including inflammation, immune response, immuno-oncology, cardiovascular II and III, and others.  The panels are run with up to about 88 samples per array (plus controls).  Because of this fixed format, these assays are priced per array, rather than per sample; meaning that they are only cost-effective when you have a sample size that is close to a multiple of 80.

Luminex Custom Order Options:

We maintain inventory of Human H80, Mouse 48, Human 48 plex.

Special order assays (charged as materials and labor): Kits can be requested, ordered and received within 1-2 weeks.  For custom kits, plan to run at least 40 samples for cost efficiency, approximately 43 duplicate samples can be run per plate. For detailed information: Please contact Yael Rosenberg Hasson-Immunoassay Manager @ yaelhr@stanford.edu or 650-724-2114 for inquiries and custom order options.

 

Custom Panel Options

Human 48 plex: https://www.emdmillipore.com/US/en/product/MILLIPLEX-MAP-Human-Cytokine-Chemokine-Growth-Factor-Panel-A-48-Plex-Premixed-Magnetic-Bead-Panel-Immunology-Multiplex-Assay,MM_NF-HCYTA-60K-PX48

Human adiponectin https://www.sigmaaldrich.com/catalog/product/mm/HADK1MAG61K?lang=en&region=US

CVD-1:

https://www.emdmillipore.com/US/en/product/MILLIPLEX-MAP-Mouse-Cardiovascular-Disease-CVD-Magnetic-Bead-Panel-1-Cardiovascular-Disease-Multiplex-Assay,MM_NF-MCVD1MAG-77K

CVD-2:

https://www.emdmillipore.com/US/en/product/MILLIPLEX-MAP-Human-Cardiovascular-Disease-CVD-Magnetic-Bead-Panel-2-Cardiovascular-Disease-Multiplex-Assay,MM_NF-HCVD2MAG-67K

CVD-3

https://www.emdmillipore.com/US/en/product/MILLIPLEX-MAP-Human-Cardiovascular-Disease-Acute-Phase-Magnetic-Bead-Panel-3-Cardiovascular-Disease-Multiplex-Assay,MM_NF-HCVD3MAG-67K

CVD-4: https://www.emdmillipore.com/US/en/product/MILLIPLEX-MAP-Human-Cardiovascular-Disease-CVD-Magnetic-Bead-Panel-4-Cardiovascular-Disease-Multiplex-Assay,MM_NF-HCVD4MAG-67K

Human immunoglobulin Isotyping Panel:

http://www.millipore.com/catalogue/item/hgammag-301k

Human Metabolic Hormone Panel:

https://www.emdmillipore.com/US/en/product/MILLIPLEX-MAP-Human-Metabolic-Hormone-Magnetic-Bead-Panel-Metabolism-Multiplex-Assay,MM_NF-HMHEMAG-34K

Human Soluble Receptor 14 Plex: 

http://www.millipore.com/catalogue/item/hscrmag32kpx14

Human Immuno-Oncology Checkpoint Panel-1

https://www.emdmillipore.com/US/en/product/MILLIPLEX-Human-Immuno-Oncology-Checkpoint-Protein-Panel-1-Immuno-Oncology-Multiplex-Assay,MM_NF-HCKP1-11K

Rat 27 plex cytokines: 

http://www.millipore.com/catalogue/item/recymag65k27pmx

User Instructions

1.  Planning your assay:  To minimize run-to-run variability, we suggest analyzing all samples in a single experiment, when possible.  To account for plate-to-plate variation and downstream analysis, it is best to evenly distribute groups across multiple plates (i.e., don't put all controls on one plate and all test samples on another) yet keep all time points per subject in the same plate.  We will run samples in the order provided, unless instructed otherwise.  Please consult 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, we request 50-70ul of plasma/serum samples. For Tissue homogenates please include a buffer with protease inhibitors, and be normalized to total protein concentration. We recommend including a control of media (for cell supernatants) or lysis buffer (for homogenates) to serve as background. Any questions please email: yaelhr@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 yaelhr@stanford.edu (or post to Basecamp for existing projects).

3. Running samples:  An online order is required before samples can be run.  We will batch samples where possible, to run complete plates.  Turnaround time varies depending on the size of your project and the current workload; Small projects can usually be completed within two weeks of order placement and sample delivery to: 1651 Page Mill Rd. room 0280.

Plasma samples are run with a 1:3 dilution due to known issues with 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.

We recommend using MFI rather than concentration values, especially for analytes that are out of the linear range of the standards.  This is usually the case for serum and plasma samples.  Normalization (to controls or to global sample median for each plate) is recommended when comparing values across plates.

Statistical consultation is available through the HIMC Statistical Consulting Service.

Protocols

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

Addtional information:

For more information

Contact:
Yael Rosenberg-Hasson, Immunoassay Manager

Address:
Human Immune Monitoring Center
1651 Page Mill Road, Lab 0280, Palo Alto, CA 94034-5518

phone:  650-723-4984
email: yaelhr@stanford.edu