The Bio-Plex system, a multiplex suspension array technique

The DKF owns a state-of-the-art multiplex suspension array system which is available for all th groups. The Bio-Plex machine is located in lab L645 on the 6 floor of the Pathology building and hosted by the group of Robert Rieben.

The Bio-Plex system is based on the xMAP technology, developed and owned by Luminex Corp., which permits multiplexing of theoretically up to 100 different ELISA-type assays within a single sample. Each assay is performed on the surface of a 5.5 m polystyrene bead. The beads are filled with different ratios of two different fluorescent dyes, resulting in an array of 100 distinct spectral address. Each set of beads can be conjugated with a different capture molecule; the conjugated beads can then be mixed and incubated with the sample in a microplate well to react with specific analytes. Capture molecules can include enzyme substrates, DNA, receptors, antigens, and antibodies. To detect and quantitate each captured analyte, a fluorescently labeled reporter molecule that specifically binds the analyte is added.

The basic principle is based on sandwich immunoassay technique. Following incubation, the contents of each microplate well are drawn into the Bio-Plex array reader, and precision fluidics align the beads in a single file through a flow cell where two lasers excite the beads individually. The red classification laser excites the dyes in each bead, identifying the specific bead address. The green reporter laser excites the reporter molecule associated with the bead, allowing quantitation of the captured analyte. Highspeed digital signal processors and the BioPlex Manager software record the fluorescent signals simultaneously for each bead, translating the signals into data for each bead-based assay.

How does the BioPlex machine look like?

Where is the Bio-Plex machine? th The Bio-Plex machine is located in the Institute of Pathology, Murtenstrasse 31, 6 floor, L645 and belongs to the DKF If you are interested to use the Bio-Plex machine, this is what you have to do: 1. Contact one of the Katja Matozan Yara Banz Robert Rieben following persons: 031 632 0586, pager 7418 031 632 0586, pager 7688 031 632 9669 katja.matozan@dkf.unibe.ch, yara.banz@dkf.unibe.ch robert.rieben@dkf.unibe.ch

2. We will arrange an introduction to the machine (30-60 min). Only persons who have had an introduction may use the Bio-Plex system! A web-calendar has been set up for reservation of the system, we details for accessing this 1 reservation system during the introduction. You should calculate at least /2 day for an experiment. Reserve online under http://www.calendar.yahoo.com Username: Password: bioplexer will be given to you during the introduction
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You should calculate at least /2 day for your experiment / measurements! How is the Bio-Plex Lab L645 organized? Available in the lab: Bio-Rad Bio-Plex workstation o array reader o microplate platform o high-throughput fluidics system o PC / monitor Calibration and Validation kit for machine calibration/validation Wash station including vacuum pump for the filter plates 2 Microtiter plate shaker Vortex Pipettes including disposable pipette tips Aluminium foil (for dark incubation)

What the user needs to bring her-/himself: 1) Everything you need to measure what you are interested in Most companies offer a kit / set of kits required for the actual test. Make sure you have all the separate ingredients you need for your test! There will be no diluents, antibodies, streptavidinPE etc. available in the Bio-Plex Lab. An example of the minimum you need for a test e.g. for cytokine measurement (Bio-Rad): 1. Cytokine multiplex panel or singleplex assay. Contains: conjugated beads, detection antibody, and lyophilized standards sufficient for 96 samples 2. Cytokine reagent kit. Includes assay buffer, wash buffer, detection antibody diluent, streptavidin-PE, 96-well filter plate, sealing tape, and instruction manual 3. Diluent kit (optional). If serum or plasma samples are to be tested, the species-specific diluent kit is used for optimum recovery

2) Your samples and everything else you need for preparation of your samples e.g. Eppendorf tubes, Falcon tubes etc.

How do I actually go about using the BioPlex machine and getting my test done? You need to go through all steps A-E regardless of the assay you are using / testing: A B C D E Remove calibration kit from fridge and leave to equilibrate to room temperature (the calibration kit will be provided as part of the infrastructure in the BioPlex lab) Power up the Bio-Plex system (2 switches [plate platform, main machine] at the back of the machine, on the right) Switch on HTF (high throughput fluidics) system (switch at the back of the machine, on the right) Switch on the PC Click onto BioPlex manager icon and follow steps according to Quick guide (pop up window on the top right) Start up (wash between plates) Calibrate* New protocol (set up your own new protocol according to plate layout) Once your layout has been set up and the parameters to be measured have been defined you can start your reading (one full plate may take up to one hour) Shutdown**

*When do I need to calibrate and why? The machine needs to be calibrated every time you use it! Calibration ensures that all system parameters are within specifications and that the signal output has been calibrated. This will ensure precision of your assay results and to make comparisons between data sets. If you have several plates to measure and a lot of time has passed between readings, the temperature of the plate reader may have gone up considerably and you may need to do a second calibration. **When do I need to perform the shutdown procedure and why? The shutdown procedure needs to be followed every time after you have finished your readings (after the final plate) and ensures that the system (needle etc.) have been rinsed thoroughly. Only after following the shutdown procedure you may follow to switch off the system! Example of a test protocol (protocol for detection of human cytokines, multiplex assay as described by Bio-Rad) If you have another kit or have set up your own test, obviously there will be slight variations to the method / dilution of standards etc. You will need the following Bio-Plex Cytokine Assay containing: - Standard, 25 g per cytokine, red labeled cover 6 - Anti-cytokine conjugated beads, 2.5x10 bead/ml, green labeled covers - Cytokine detection antibody (single-plex, 8-plex or 17-plex) blue labeled covers Bio-Plex Reagent Kit A containing: - Bio-Plex assay buffer A - Bio-Plex wash buffer A - Bio-Plex detection antibody diluent A - Streptavidin-PE (100 x) - Sterile 96-well filter plate with cover and tray, sealing tapes Bio-Plex Diluent Kit A containing: - Bio-Plex sample diluent A - Bio-Plex standard diluent A 1. Set up a plate layout (eg. on paper, easier to calculate needed dilutions!) 2. Preparation of Standard Curve - reconstitute all cytokine standards in 50 l cold sterile water (concentration of stock solution: 500000 pg/ml) - leave to rest for 30 min on ice - add 450 l standard diluent (for serum/plasma samples) or cell culture medium (for cell culture supernatant) to the standard stock solution - prepare serial dilution of the standards as shown below (a minimum of 6 standards should be used)

3. Sample preparation - dilute samples in Bio-Plex sample diluent, 1:4 (or as required e.g. serum, plasma) or use neat (e.g. cell culture supernatant), around 6 0l is sufficient (effectively 50 l per well) 4. Bead stock and plate preparation - pre-wet the plate with 100 l assay buffer A per well (buffer for human samples) - dilute anti-cytokine conjugated beads 1:25. Per well: 2 l beads + 48 l buffer A to calculate total amount of bead solution needed for the wells: no. of wells x 50 l (+ 100 l for every 400 l to be on the safe side) - transfer 50 l anti-cytokine conjugated beads into the plate - leave beads to settle down a little - add 100 l wash buffer A to each well, place plate on wash platform, switch on vacuum pump to remove excess fluid - release vacuum and gently ease plate off wash platform - again add 100 l wash buffer A to each well, place plate on wash platform and remove excess fluid. (Take care not to remove excess fluid too violently so that beads dont get pulled into plate filter membrane!) 5. Incubation: samples with anti-cytokine conjugated beads - add 50 l of standards/samples per well - place plate on the plate shaker (see below), fix corner with elastic band, for 30 sec at 1100 rpm, followed by 30-60 min at 300 rpm, room temperature - wash 3 times with wash buffer A, 100 l as above 6. Incubation: anti-cytokine detection antibody - dilute the antibody to 1 x concentration in antibody diluent (from stock solution of 100 x, 50 x or 25 x, check on the tube!) - add 25 l diluted antibody solution per well - place plate on the plate shaker, fix corner with elastic band, for 30 sec at 1100 rpm, followed by 30-60 min at 300 rpm, room temperature - wash 3 times with wash buffer A, 100 l 7. Incubation: Streptavidin-PE - dilute the Streptavidin-PE solution to 1 x concentration (from stock solution of 100 x) in assay buffer A to 1x concentration - 50 l per well is needed, calculate at least 2 extra wells for every 8 wells - incubate on the plate shaker, 30 sec @ 1100 rpm then 10-30 min @ 300 rpm - wash 3 times with wash buffer A, 100 l 8. Prepare plate for read out - pipet 100 l assay buffer A into each well containing the beads for plate reading plate is now ready to be put into Bio-Plex machine for read out!

Links to the Luminex web page and some selected companies offering xMAP products Via the Luminex web page you can find links to most other companies offering products to use in a Luminex system http://www.luminexcorp.com/01_xMAPTechnology/03_partners.html Bio-Rad beads, complete cytokine-kits for human, mouse and rat; phosphoprotein-kits, diluents, and bead coupling-kits reagents http://www.bio-rad.com R&D Systems beads, reagents, diluents, MMP-panels, cytokine-kits, diluents http://www.rndsystems.com/asp/g_sitebuilder.asp?BodyId=495 Qiagen carboxy beads for coupling http://www1.qiagen.com/products/protein/MultiplexBeadBasedAssays.aspx Upstate cytokine, chemokine, growth factor and protein detection kits; serum diluent kits, for human and mouse, kinase activity assays, immunoglobulins http://www.upstate.com/browse/categories/beadlyte.asp?c=25&r=71 Biosource cytokines, chemokines, inflammatory markers, death receptors, growth factors: 36 human, 22 mouse parameters http://www.biosource.com/content/catalogContent/prodcatlist.asp Labodia Distributor for LINCO Research Products. Cytokines, chemokines, cardiovascular disease markers, endocrine markers, apolipoproteins, sepsis and apoptosis markers http://www.labodia.com