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 Array or Olink Arrays which utilizes PEA Technology. Biomarker information in our standard Luminex kits can be downloaded 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.

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Luminex Human 80 plex (EMD-Millipore)

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

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 Yael Rosenberg Hasson: Immunoassay Manager @ yaelhr@stanford.edu or 650-724-2114.

 

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 yaelhr@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: 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 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, 650-7234984.

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.

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.

Additional information:

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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