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FNN0021

Product FAQ

How do I process oral mucosal transudate for Luminex™ assays?

Answer

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.

Answer Id: E12649

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Product FAQ

How do I process saliva samples for the Luminex™ assay platform?

Answer

We have not specifically tested saliva samples in-house. 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 treat the sample as you would a supernatant; follow the procedure described for cell culture supernatant.

Answer Id: E12650

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Product FAQ

How do I process serum samples for use on the Luminex™ assay platform?

Answer

Serum samples should be collected in pyrogen/endotoxin-free tubes. Whole blood should be allowed to sit at room temperature for 15-30 min to clot. Spin at 1,000-2,000 x g for 10 min in a 4 degrees C refrigerated centrifuge to separate the cells. 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 serum is to be analyzed at a later date, apportion it into 0.5 mL aliquots and store at -80 degrees C. Avoid multiple freeze-thaw cycles. When possible, avoid the use of hemolyzed or lipemic sera. We recommend that upon thawing the samples be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered, prior to analysis, to prevent clogging of the filter plates and/or probe. Follow the assay procedure provided with the kit for appropriate dilutions.

Answer Id: E12640

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Product FAQ

Can I store Luminex™ assay plates overnight and read them the next day?

Answer

If the plate cannot be read on the day of the assay, cover and store the plate in the dark overnight at 2-8 degrees C for reading the following day without significant loss of fluorescence intensity. When you are ready to read the plate, bring the plate to room temperature on an orbital plate shaker (still protected from light). Aspirate the Working Wash Solution from the stored plates and add 100 μL of fresh Working Wash Solution. Place the plate on an orbital shaker for 2-3 min at 500-600 rpm prior to analysis.

Answer Id: E12653

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Product FAQ

I am getting a warning message that there is high bead aggregation. What should I do?

Answer

Here are possible causes and solutions for this issue:

Insufficient bead mixing by sonication and vortexing: Vortex the beads for at least 30 seconds, and then sonicate the beads for at least 30 seconds before adding them to the assay. Be sure your plate shaker is set appropriately for optimal bead movement in the wells during the incubation steps.
Entry of air into the line of the Luminex™ instrument, due to insufficient bead suspension volume in the well for the height of the instrument probe: Verify that the beads are suspended in an appropriate amount of solution for analysis (see assay specific instructions). Adjust the sample probe height.

Answer Id: E12663

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Product FAQ

How do I process synovial fluid on the Luminex™ assay platform?

Answer

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.

Answer Id: E12645

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Product FAQ

The bead count for my standard curve is correct and regular, but for my samples the bead count is erratic and I get a warning message saying “The acquisition had at least one region that did not reach the maximum count”. What should I do?

Answer

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.
Confirm that the reagents appropriate for your sample type have been used.
Confirm that there is at least a 1:1 ratio of sample to assay diluent for serum, plasma, CSF, and culture supernatant samples. For cell lysates or tissue homogenates, confirm that the sample has been diluted appropriately to reduce the concentration of detergent from the lysis buffer to ?0.01%. For other sample types, further sample optimization may be required.

Answer Id: E12659

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Product FAQ

I bought one of your Antibody Pair ELISA kits, but there were no buffers included. How should I set up and run the ELISA?

Answer

Our Antibody Pair kits include matched, pre-titered and fully optimized coating and detection antibodies, 3 vials of recombinant protein standard, the streptavidin-HRP conjugate concentrate, and a lot-specific technical data sheet. The buffers are not included. The Buffer Kit needed to run the Antibody Pair ELISA must be purchased separately (Cat. No. CNB0011). Note that the 5X Assay Buffer supplied in Cat. No. CNB0011 is used to block your ELISA plates and as a diluent for the standards and samples.

Answer Id: E12627

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Product FAQ

How do I develop a sandwich ELISA using Antibody Pairs?

Answer

Each Antibody Pair kit contains capture (coating) antibody, biotinylated detection antibody, recombinant standard, and streptavidin-HRP. Other reagents required are listed in the Antibody Pair manual included with the kit, and can also be purchased separately (Antibody Pair Buffer Kit, Cat. No. CNB0011; 5X Assay Buffer, Cat. No. DS98200; etc.). The manual also provides a specific procedure and illustrates an example of a standard curve that can be obtained when the specific procedure is followed.

A general procedure is summarized here:
1) Coat the microplate with diluted capture (coating) antibody overnight at 2-8 degrees C; wash the plate.
2) Incubate the standards or samples in the coated microplate; wash the plate.
3) Incubate diluted biotinlyated detection antibody in the plate; wash the plate.
4) Incubate streptavidin-HRP in the plate for 15-45 min; wash the plate.
5) Incubate the plate with TMB substrate for 10-60 min, and then stop the reaction with Stop solution.
6) Read the microplate at 450 nm.
We recommend determining optimal buffer formulations, concentrations, and incubation times for individual applications.

Answer Id: E12616

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Product FAQ

How do I process cell lysates (extract cellular proteins) for the Luminex™ assay platform?

Answer

The protocols mentioned below have been applied to several human and mouse cell lines. You should optimize the cell extraction procedures for your own applications.

Cell Lysis Procedure

Non-adherent cells:
Pellet cells by low-speed centrifugation. Remove the medium from the pellet, and wash twice with ice-cold PBS. Remove the PBS, and resuspend the cell pellet in cell lysis buffer (recommended cell lysate concentration is 2-5 mg/mL). Incubate 15 min on ice with occasional vortexing. Transfer the lysate to a microcentrifuge tube and centrifuge at 14,000 rpm for 10 min at 2-8 degrees C. Aliquot the cleared lysate into clean microcentrifuge tubes, and determine total protein concentration.

Adherent cells:
Remove the tissue culture medium from the cells, and wash twice with ice-cold PBS. Remove the PBS, add cell lysis buffer (recommended cell lysate concentration is 2-5 μg/mL), and incubate 15 min on ice. Collect the cell lysate and transfer it to a microcentrifuge tube. Centrifuge at 14,000 rpm for 10 min at 2-8 degrees C. Aliquot the cleared lysate into clean microcentrifuge tubes, and determine total protein concentration. Lysates should be frozen and stored at -80 degrees C or analyzed shortly after collection. Avoid multiple freeze-thaw cycles of frozen samples. Thaw completely, mix well, and clarify by centrifugation (14,000 rpm for 5 min) prior to analysis to prevent clogging of the filter plates.

Recommended Cell Lysis Buffer:
NP40 Cell Lysis Buffer (Cat. No. FNN0021) Note: Lysates prepared with NP40 Lysis Buffer must be diluted at least 5-fold prior to running the assay. The recipe to make the buffer is as follows: 50 mM Tris, pH 7.4, 50 mM NaF, 260 mM NaCl, 1 mM Na3VO4, 5 mM EDTA, 0.02% NaN3, 1% Nonidet P40. The NP40 Cell Lysis Buffer (without protease inhibitor cocktail and PMSF) is stable for 2-3 weeks at 2-8 degrees C, or for 6 months when stored in aliquots at -20 degrees C.

Add fresh to the NP40 Cell Lysis Buffer just before use:
1 mM PMSF (stock 0.3 M in DMSO)
Protease inhibitor cocktail (Sigma, Cat. No. P-2714)
Alternate Cell Extraction Buffer
Cell Extraction Buffer (Cat. No. FNN0011) Note: Lysates prepared with Cell Extraction Buffer must be diluted at least 10-fold prior to running the assay. Or 10 mM Tris, pH 7.4 2 mM Na3VO4 100 mM NaCl 1% Triton X-100 1 mM EDTA 10% glycerol 1 mM EGTA 0.1% SDS 1 mM NaF 0.5% deoxycholate 20 mM Na4P2O7 The Cell Extraction Buffer (without protease inhibitor cocktail and PMSF) is stable for 2-3 weeks at 2-8 degrees C, or for 6 months when stored in aliquots at -20 degrees C. Add fresh to the Cell Extraction Buffer just before use: 1 mM PMSF (stock 0.3 M in DMSO) Protease inhibitor cocktail (Sigma, Cat. No. P-2714)

Answer Id: E12651

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Product FAQ

How do I process tissue homogenates for use on the Luminex™ assay platform?

Answer

We recommend following the protocol provided below, which was developed using the Tissue Extraction Reagent I (Cat. No. FNN0071), and shows good correlation between ELISA and Luminex™ technology. This procedure has been applied to multiple tissue types. However, we recommend that you optimize for each tissue sample type used. Similar extraction reagents/lysis buffers may be used.

Add protease inhibitors to the Tissue Extraction Reagent just before use.
Weigh the tissue sample.
Add 10 mL of the Tissue Extraction Reagent per 1 gram of tissue.
Homogenize the tissue.
Centrifuge the sample at 10,000 rpm for 5 min to pellet the tissue debris.
Collect the supernatant. Follow the assay procedure provided with the kit for appropriate dilutions; this is to prevent/minimize potential inhibition of antibody-antigen binding by the detergent present in the extraction or lysis buffer. In general, tissue homogenates or cell lysates (depending on the lysis buffer used) need to be diluted 5- to 10-fold to reduce the detergent concentration to ?0.01%. However individual kits/samples may require further dilution with assay diluent or standard diluent based on the concentration of the cytokine/protein of interest.
Note: If the samples are to be stored, aliquot them and freeze at -80 degrees C. Avoid multiple freeze-thaw cycles.

Answer Id: E12643

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Product FAQ

My sample lysates were made in RIPA buffer. Can I test these samples with your ELISA kits?

Answer

Yes, you can. The composition of the traditional 1X RIPA buffer is very similar to that of our Cell Extraction Buffer (Cat. No. FNN0011). Cat. No. FNN0011 is frequently used to prepare lysates for testing with our ELISA and Luminex™ kits. Our NP-40-based Cell Extraction Buffer (Cat. No. FNN0021) is also used. We recommend diluting lysates made with Cat. No. FNN0011 at least 10-fold in order to lower the SDS concentration to less than or equal to 0.01% (v/v) before adding the samples to the ELISA or Luminex™ assay.

Answer Id: E12625

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Product FAQ

How do I process plasma samples for use on the Luminex™ assay platform?

Answer

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.

Answer Id: E12641

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Product FAQ

My filter plate is leaking. What should I do?

Answer

Here are some possible causes and solutions for this problem:

Placing the plate on an absorbent material both at the bench and on the shaker (which results in wicking when there is contact between the absorbent material and the opening on the bottom of the well funnel). Rest the plate currently in use on top of a second filter plate so that the filter plate currently in use only comes into contact with a nonabsorbent surface. This will also help locate any potential leaks.
Prior to incubations, use a Kimwipe™ tissue and lightly press up on each well to dry off the bottom of the plate. Putting any vertical pressure on the plate during the incubations (using either tape or clamps).
Use clamps that fit around the sides of the filter plate to secure the plate to the shaker during incubations. Subjecting the plate to vacuum pressure greater than 5 mm Hg, even for a moment, which either tears the filter membrane or creates an opening that exceeds the design limits for volume retention.
Prevent any vacuum surge by opening and adjusting the vacuum on the manifold before placing the plate on the manifold surface and checking the vacuum with a test plate (i.e., not the plate used for the assay). For our assays we recommend the setting not exceed 5 mm Hg. Puncturing the filter membrane with pipet tips, by inserting the tips all the way into the wells during reagent loading.
Pipet solutions along the sides of the wells, rather than deep into the wells. Separating the soft (opaque) plastic bottom from the hard (clear) plastic top of the filter plate before starting, which compromises the integrity of the seal even if the pieces appear to fit back together properly. If you accidentally separate these two layers of the plate, the plate should be discarded and a fresh plate used instead.

Answer Id: E12654

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Product FAQ

How do I process cerebrospinal fluid (CSF) for use on the Luminex™ assay platform?

Answer

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. However, CSF has relatively low viscosity, and unless there is presence of an infected state (abundance of WBCs), it should not require clarification and can be directly applied to the assay. Dilute 2-fold with Assay Diluent provided in the Neuroscience Buffer Kit (Cat. No. LNB0001).

Answer Id: E12646

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