Luminex® Performance Assay Human Cytokine Base Kit A Catalog Number LUHM000 For the simultaneous quantitative determination of multiple human cytokine concentrations in cell culture supernates, serum, and plasma. This package insert must be read in its entirety before using this product. For research use only. Not for use in diagnostic procedures.

TABLE OF CONTENTS SECTION Manufactured and Distributed by: USA R&D Systems, Inc. 614 McKinley Place NE, Minneapolis, MN 55413 TEL: 800 343 7475 612 379 2956 FAX: 612 656 4400 E-MAIL: [email protected] Distributed by: Europe | Middle East | Africa Bio-Techne Ltd. 19 Barton Lane, Abingdon Science Park Abingdon OX14 3NB, UK TEL: +44 (0)1235 529449 FAX: +44 (0)1235 533420 E-MAIL: [email protected] China Bio-Techne China Co., Ltd. Unit 1901, Tower 3, Raffles City Changning Office, 1193 Changning Road, Shanghai PRC 200051 TEL: +86 (21) 52380373 (400) 821-3475 FAX: +86 (21) 52371001 E-MAIL:...

INTRODUCTION Cytokines are intercellular signaling proteins released from a wide variety of cells and tissues. They play an integral role in regulating growth and cellular proliferation as well as modulating host response to infection, injury, and inflammation. Cytokines also influence reproduction and bone remodeling. A large number of cytokines are pleiotropic and share similar functions. In addition, many cytokines influence the production of other cytokines. Analysis and quantification of cytokines in biological fluids and cell culture supernates has thus become increasingly important. Methods...

PRINCIPLE OF THE ASSAY Luminex® Performance Assay multiplex kits are designed for use with any Luminex analyzer including the MAGPIX®, Luminex® 100/200™, FLEXMAP 3D®, xMAP INTELLIFLEX®, or Bio-Rad® Bio-Plex®, dual laser, flow-based sorting and detection platforms. Analyte-specific antibodies are pre-coated onto magnetic microparticles embedded with fluorophores at set ratios for each unique microparticle region. Microparticles, standards and samples are pipetted into wells and the immobilized antibodies bind the analytes of interest. After washing away any unbound substances, a biotinylated antibody...

Store the unopened kit at 2-8 °C. Do not use past kit expiration date. ^Provided this is within the expiration date of the kit. Note: Additional wash buffer is available for purchase (R&D Systems®, # WA126).

OTHER SUPPLIES REQUIRED • Luminex® Performance Assay analyte-specific kit(s) (see page 1) • MAGPIX®, Luminex® 100/200™, FLEXMAP 3D®, xMAP INTELLIFLEX®, or Bio-Rad® Bio-Plex® analyzer with X-Y platform • Hand-held microplate magnet or platewasher with a magnetic platform • Pipettes and pipette tips • Deionized or distilled water • Multi-channel pipette, manifold dispenser, or automated dispensing unit • 50 mL and 500 mL graduated cylinders • Horizontal orbital microplate shaker (0.12" orbit) capable of maintaining a speed of 800 ± 50 rpm • Microcentrifuge • Polypropylene test tubes for dilution...

SAMPLE COLLECTION & STORAGE The sample collection and storage conditions listed below are intended as general guidelines. Sample stability has not been evaluated. Cell Culture Supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes at room temperature before centrifuging for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles. Plasma - Collect...

REAGENT PREPARATION Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Add 20 mL of Wash Buffer Concentrate to 480 mL of deionized or distilled water to prepare 500 mL of Wash Buffer. Calibrator Diluent RD5K (diluted 1:2) - For cell culture supernate samples only. Add 20 mL of Calibrator Diluent RD5K to 20 mL of deionized or distilled water to prepare 40 mL of Calibrator Diluent RD5K (diluted 1:2). Standards - Refer to the Standard Value Cards for the...

1. Centrifuge each Microparticle Concentrate vial for 30 seconds at 1000 x g prior to removing the cap. 2. Gently vortex the vials to resuspend the microparticles, taking precautions not to invert the vials. 3. Dilute the Microparticle Concentrates in the mixing bottle provided. The volume of the Microparticle Concentrate listed in the table below is for each analyte (e.g. if measuring a full plate of IL-ip and IL-6, add 50 pL of IL-1p Microparticle Concentrate and 50 pL of IL-6 Microparticle Concentrate to 5 mL of Microparticle Diluent).

INSTRUMENT SETTINGS Note: Adjust the probe height setting on the analyzer to avoid puncturing the plate. Calibrate the analyzer using the proper reagents for superparamagnetic microparticles (refer to instrument manual). MAGPIX® analyzer: a) Sample volume: 50 μL b) Assign the microparticle region for each analyte being measured (see page 1) c) 50 count/region d) Collect Median Fluorescence Intensity (MFI) Luminex® 100/200TM, FLEXMAP 3D®, xMAP INTELLIFLEX®, and Bio-Rad® Bio-Plex® analyzers: Note: Ensure that the instrument flow rate is set to the default of 60 μL/minute (fast) for all flow based...

ASSAY PROCEDURE Bring all reagents and samples to room temperature before use. It is recommended that all standards, controls, and samples be assayed in duplicate. Note: Protect microparticles and Streptavidin-PE from light at all times. 1. Prepare all reagents, working standards, and samples as directed in the previous sections. 2. Add 50 μL of standard, control, or sample* per well. A plate layout is provided to record standards and samples assayed. 3. Resuspend the diluted Microparticle Cocktail by inversion or vortexing. Add 50 μL of the microparticle cocktail to each well of the microplate....