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View additional product information for Luminex 200™ Performance Verification Kit - FAQs (LX2RPVERK25)
20 product FAQs found
Here are possible causes and solutions for this issue:
- Check the protocol settings (make sure you select the correct DD settings).
- Check the level of sheath fluid and empty the waste.
- Before acquiring the plate, run calibration and verification beads on the Luminex instrument.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are possible causes and solutions for this issue:
This usually indicates that the beads have been photobleached. This problem can also be caused by exposing the beads to organic solvents. Unfortunately, the assay will have to be repeated because the beads cannot be restored. The beads must be protected from light and organic solvents.
Alternatively, the instrument may be off in its measurements or you may have a calibration issue. Call the manufacturer for a service appointment.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
This indicates that an incorrect buffer was used for the final step. The Wash Solution provided in the kit must be used for washing the beads and the Reading Buffer should be used for resuspending the beads before loading them into the Luminex instrument. The osmolarity of the solution will impact the size of the bead, and any change in the bead size will alter detection by the instrument.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are some suggestions:
- Before acquiring the plate, run calibration and verification beads on the Luminex instrument.
- Review the instrument settings and make sure they are appropriate for the assay being run (adjustment of needle height, make sure you select the correct bead gates and the correct DD settings).
- Shake the plate before acquisition on the instrument to resuspend the beads.
- Vortex the beads for 30 sec before adding them into the plate.
- Washing: Do not forget to keep the plate for about 2 mins on the Hand-Held Magnetic Plate Washer before emptying the plate.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
This pattern is indicative of a sample matrix effect. Here are some suggestions:
- Confirm that the sample has been clarified and is free of debris and free of lipids (5-10 min centrifugation recommended).
- Confirm that there is at least a 1:1 ratio of sample to assay diluent for serum, plasma samples. For cell lysates or tissue homogenates, confirm that the sample has been diluted appropriately in assay buffer to reduce the concentration of detergent in the lysis buffer to ⋜0.01%. For other sample types, further sample optimization may be required.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are some suggestions:
- It is possible that the levels of your protein of interest fall below the detection limits of the assay. High Sensitivity Multiplex kits are available for most cytokines.
- Qualify your standard curve (look for plateaus, abnormal curve fits, outliers).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Here are some suggestions:
- Sample optimization may be needed: Dilute the sample with an appropriate diluent and re-run.
- Qualify your standard curve (look for plateaus, abnormal curve fits, outliers).
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
To process cell lysates (extract cellular proteins), follow the instructions provided here (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017835_PrepareCellLysates_Procartaplex_PI.pdf).
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We have not specifically tested saliva samples in-house and therefore these instructions are only our recommendation. Saliva contains several proteolytic enzymes. It would be important to centrifuge samples and be sure not to pipet any cellular material or debris into the assay plate. We would suggest adding some anti-protease in the sample (for example, trasylol or aprotinine, 10 to 50 U/mL) to protect the protein from enzyme degradation. You may then centrifuge the samples at 1000 x g at 4 degrees C for 10 mins to remove particulates. Use immediately or store aliquots at -80 degrees C. Avoid multiple freeze-thaw cycles.
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We have not specifically tested oral mucosal transudate samples in-house and therefore these instructions are only our recommendation. Isolate the site around the tooth and insert a piece of periodontal filter paper into the gum pocket around the tooth for 30 seconds. Remove the filter paper and extract 4 times with 50 µL PBS for 5 min each at room temperature. The individual extractions can be combined and analyzed. Dilute 2-fold with Assay Diluent before applying to the assay.
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We have not specifically tested cervical secretion samples in-house and therefore these instructions are only our recommendation. Cervical sponges should be placed on ice immediately upon collection. Samples should be stored at -20 degrees C for up to one week and then stored at -80 degrees C until ready for assay. After thawing, sponges should be weighed and placed into Eppendorf tubes, using forceps cleaned with ethanol after each transfer. Add 200 µL of ice-cold extraction buffer (recipe below) to each tube and incubate overnight at 4 degrees C. The sponges and extraction buffer can then be transferred to microcentrifuge tubes with 0.2 µm cellulose acetate filters and centrifuged at 13,000 rpm for 10 min at 4 degrees C. The eluate can then be tested for cytokine expression.
Extraction Buffer
50 mM HEPES, pH 7.5
1 mM Na3VO4
150 mM NaCl
1 mM NaF
1 mM EDTA
0.1% Tween 20
25 mM EGTA
10% glycerol
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We have not specifically tested bronchoalveolar lavage (BAL) samples in-house and therefore these instructions are only our recommendation. The bronchoalveolar lavage (BAL) should be collected in a sterile syringe and kept on ice until you are ready to analyze it. Alternatively, BAL can be aliquoted and frozen in usable sample sizes (such that exposure to freeze-thaw is limited to one time). All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute 2-fold with Assay Diluent before applying to the plate.
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Centrifuge samples at 1,400 rpm for 10 mins at 4 degrees C to remove particulates. Use immediately or store aliquots at -80 degrees C. Avoid multiple freeze-thaw cycles.
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We have not specifically tested synovial fluid samples in-house and therefore these instructions are only our recommendation. Collect synovial fluid into non-heparinized tubes and spin at 1,000 x g for 10 min within 30 min of sample collection. The acellular portion of synovial fluid should be stored at -80 degrees C before subsequent analysis. All samples need to be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered prior to analysis to prevent clogging of the filter plates. Dilute samples 1:1 with Assay Diluent prior to addition to the assay. Reference: Raza K et al. (2005) Arthritis Research &Therapy 7(4): R784-R795.
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Collect spleen, lung, brain, kidney, liver, or heart tissue and treat with or without LPS (100 µg, i.p., for 15 mins, 30 mins, 1 hr, 2 hrs, or 3 hrs). Weigh tissue in a 2 mL microcentrifuge tube. Add 500 µL of Cell Lysis Buffer (Cat. No. EPX-99999-000) per 100 mg of tissue. Add one 5-mm Stainless Steel Bead, then assemble tubes into TissueLyser according to the manufacturer’s recommendations. We recommend using 5-mm Stainless Steel Beads from Qiagen (Cat. No. 69989). Homogenize tissue at 25 Hz for 0.5-3 mins as indicated in the table below. Centrifuge the sample at 16,000 × g for 10 mins at 4 degrees C. Transfer the supernatant to a new microcentrifuge tube. Measure the total protein concentration. Dilute samples to 10 mg protein/mL with 1X PBS. To proceed with ProcartaPlex protocol, add 25 µL of Universal Assay Buffer (Cat. No. EPX-11111-000) to 25 µL of the diluted sample to each sample well.
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Cells should be in log-phase growth. Stimulate cells as desired in appropriate cell culture flasks. Using sterile technique, remove the desired volume of conditioned cell culture medium with a pipette and transfer the medium to clean polypropylene microcentrifuge tubes. Centrifuge the medium at 14,000 rpm for 10 min at 4 degrees C in a refrigerated microcentrifuge to remove any cells or cellular debris. Aliquot the clarified medium into clean polypropylene microcentrifuge tubes. These samples are ready for the assay. Alternatively, clarified medium samples can be aliquoted and stored at -80 degrees C for future analysis. Avoid multiple freeze-thaw cycles. Frozen samples should be allowed to thaw on ice just prior to running the assay. Thawed samples should be clarified by centrifuging at 14,000 rpm for 10 min at 4 degrees C in a refrigerated microcentrifuge prior to analysis to prevent clogging of the Luminex probe and/or filter plate. Follow the assay procedure provided with the kit for appropriate dilutions.
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Separate the cells from the plasma samples by centrifugation at 2,000 x g for 10 min in a refrigerated centrifuge. Centrifugation at this force is necessary to deplete the sample of platelets. Transfer the supernatant to a chilled clean polypropylene tube with a sterile Pasteur pipette. Maintain the samples at 2-8 degrees C while handling.
If the plasma is to be analyzed at a later date, apportion it into aliquots in polypropylene microcentrifuge tubes and store at -80 degrees C. Avoid multiple freeze-thaw cycles. When you are ready to analyze them, allow the samples to thaw on ice. All plasma samples should be clarified by centrifugation (14,000 rpm for 10 min at 4 degrees C) in a refrigerated microcentrifuge immediately prior to analysis. Follow the assay procedure provided with the kit for appropriate dilutions.
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Serum samples should be collected in pyrogen/endotoxin-free tubes. Whole blood should be allowed to clot for 20-30 mins at 20-25°C. Centrifuge at 1,000 x g for 10 mins at 20-25°C and collect the serum fraction. Alternatively, a serum separator tube can be used following the manufacturer's instructions. Use immediately or store aliquots at -80°C. Avoid multiple freeze-thaw cycles.
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Luminex xMAP technology is based on polystyrene or paramagnetic microspheres, or beads, that are internally dyed with red and infrared fluorophores of differing intensities. Each dyed bead is given a unique number, known as a “bead region”, allowing the differentiation of beads. For ProcartaPlex multiplex immunoassay kits, individual bead sets are then coated with a capture antibody qualified for one specific analyte. Multiple analyte-specific beads can then be combined in a single well of a 96-well assay to detect and quantify multiple targets simultaneously, using one of the Luminex instruments for analysis.
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The Luminex assay is a bead-based immunoassay that uses beads of defined spectral properties conjugated to protein-specific capture antibodies and added along with samples (including standards of known protein concentration and test samples) into the wells of a microplate. The target protein binds to the capture antibodies over the course of a 2 hr incubation. After incubation on a shaker, the beads are washed by putting the 96-well plate on a flat magnet for 30 seconds, after which the fluid is discarded by flicking the wells or by using an automated plate washer. The magnet is removed, and the beads are resuspended in the detection antibody. Another incubation and wash are followed by the addition of streptavidin–R-phycoerythrin (SAPE). The beads are then washed and are ready to analyze. The Luminex technology is compatible with the following Luminex analyzers:
MAGPIX System-affordable, efficient, and compact
Luminex 200 System-versatile, efficient, and widely used in multiplexing
FLEXMAP 3D System-high throughput (up to 500 simultaneous assays) and automation compatible
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.