Multiplex Analysis Core

• Download the authorization form (DOC)
• E-mail the completed form to Kristina Taylor at kristtay@iu.edu and the MAC at pplett@iu.edu

Find an off-the-shelf kit or create a custom kit that will identify your specific targets, tissue, and species. Some resources include:

• xMAP kit finder (Select Luminex 100/200 instrument)
• Assay Builder website for Bio-Rad bio-plex products
• Published literature
• Technical support from your favorite company (see commercial contacts below)

• Each kit contains enough reagents for 96 reactions. Standards and blanks consume 18-20 reactions, leaving enough reagents for 76-78 samples run in singlet, or 38-39 samples run in duplicate, as you prefer, although this can vary slightly depending on number of standards and/or inclusion of quality control samples as per vendor.      
• Collect samples following information in the “Technical Procedures” section below.
• Additional instructions on sample preparation are included in kits, or on the vendor websites (see contacts).

• Request the requisition form from the MAC (pplett@iu.edu or hfeng@iu.edu). 
• The MAC will contact you to schedule your assay time.
• Submit the requisition form at least 24 hours before your scheduled run.
• If you need to cancel or postpone your assay time the day of the run, please call 317-278-2485 the MAC by 9 a.m. on the day of analysis to avoid being charged for instrument set-up.

All data generated by the MAC are kept confidential and will not be shared unless the owner provides specific written direction.

• Payment is expected for services rendered. An IU account number or a purchase order number (PO#) must be provided before service is provided, and the PI or person responsible for payment must sign and date the authorization form.
• Full payment is expected if data are obtained regardless whether or not it represents the result expected by the investigator.
• Partial or waived payments may be accepted in cases of instrument malfunction.
• The MAC does not provide sample storage after analyses. Samples not retrieved from the MAC within one week of analyses will be discarded.

Work is prioritized based on a first-come, first-served policy.

Authorship is not expected when routine analyses are performed by the Multiplex Analysis Core. However, if effort from core personnel exceeds that of routine analyses, authorship will be discussed and agreed upon prior to running the users’ samples.

If research supported by the MAC results in publication, please acknowledge this support by including the following in your publication(s): We thank the Multiplex Analysis Core at the Indiana University Melvin and Bren Simon Comprehensive Cancer Center for providing support in analyzing samples and interpretation of data.

Please notify the MAC of your publication by emailing us at corschel@iu.edu or pplett@iu.edu.

The director will discuss all issues with the investigator in order to resolve them. Should issues (such as charge disputes, authorship, and conflict of interest) arise that cannot be resolved in this manner, the Advisory Committee will assist in conflict resolution.

• Material or health-related information submitted to a Core for analysis must be obtained in compliance with the most current regulatory and legal requirements (including material transfer agreements).
• It is the responsibility of the investigator to make sure such requirements are fulfilled.
• It is also the responsibility of the investigator to comply with all safety standards (e.g. radiation and biosafety) and fully disclose the potential hazards of all materials submitted to the core.

• For information on collecting serum versus plasma for your cytokine analyses, see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4332596/ When heparin is used as the anticoagulant in plasma samples, please check your kit manual for the proper heparin concentration (Millipore suggests using <10 IU/mL Heparin in the plasma).Minimize hemolysis as much as possible as iron may interfere with some analyses.
• Store samples in small aliquots to avoid freeze thawing if running multiple assays. Store approximately 50ul per single well in the Bioplex assay, or ~100µL if duplicate wells will be run. Note that the volume required will depend on which analyte is assayed, as some require dilution before being assayed.
• On your scheduled analysis day, bring your samples on dry ice and kits on wet ice to the MAC.
• To ensure that the samples are free of particulates, we will centrifuge the samples at 10,000 x g on a benchtop microcentrifuge for 10 minutes.

• Centrifuge samples at 1,000-1,200 x g for 10 min at 4ºC. Harvest supernatant.
• Store samples in aliquots of 75µL each (for singlet analysis) or 150µL (for duplicate analyses) at -70ºC.
• On your scheduled analysis day, bring your samples on dry ice and kits on wet ice to the MAC, along with tissue culture medium (~1ml) for dilution of standards. NOTE: There is usually no need to dilute tissue culture samples, but if dilution is necessary, dilute with tissue culture media to achieve a final volume of at least 75 µl if analyzing in singlet or 150 µl if analyzing in duplicate.
• To ensure that the samples are free of particulates, we will centrifuge the samples at 10,000 x g on a refrigerated benchtop microcentrifuge for 10 minutes.

• Protocols for preparation of cell lysates from specific tissues are numerous and can be found on vendor websites and in publications.
• The investigator is encouraged to use any protocol he or she feels is best for their particular application. A tissue lysate protocol is also included in the Bio-Rad kit for phosphoproteins, which may also be used for cytokine analyses of tissue lysates.
• Additional sample preparation protocols can be found on the vendor websites listed below.

Effective: July 1, 2024

Add $14/ plate for kits requiring a dilution step.

The IU Simon Comprehensive Cancer Center will invoice the investigator when the work is completed using the account number provided on the authorization form.

The Bio-Plex 200 Multiplex System with High Throughput Fluidics (HTF) is a suspension bead-based bioassay system that uses microscopic beads dyed with different ratios of two fluorochromes that emit a color spectrum unique to that fluorochrome ratio.

Each bead is also tagged with an antibody specific to a certain target. Up to 100 differentially dyed beads can be used in a single multiplex assay, each conjugated to a different antibody. These bead-antibody conjugates are incubated with the target sample, washed, and then incubated with biotin-labeled detection antibodies (BIOT/Ab) specific for secondary epitopes on the target.

Samples are washed and then incubated with a phycoerythrin (PE) labeled streptavidin reporter (SA/PE), washed, and resuspended for analysis.

The fluorescence of the bead-antibody-target-BIOT/Ab-SA/PE complex is then read on the Bio-Plex by a red laser that identifies the specific bead by its fluorescent signature and a green laser that excites the PE dye, with the intensity of PE fluorescence signal indicating the level of target bound.