ThermoFisher SCIENTIFIC MAN0028348 Human Simplex and Combinable Panels

Specifications

• Product Name: ProcartaPlexTM Human Simplex and Combinable Panels
• Optimized for: Detection of multiple analytes from serum, plasma, and cell culture supernatants
• Validated for: Urine, cell lysates, or cerebrospinal fluid (CSF) samples

Product Information

The ProcartaPlexTM Human Simplex and Combinable Panels are designed for detecting multiple analytes in various sample types including serum, plasma, and cell culture supernatants. The assays have also been validated for use with urine, cell lysates, or cerebrospinal fluid (CSF) samples.

For detailed product information, visit thermofisher.com/procartaplex.

Contents and Storage

• Components Supplied:
  • Antigen Standards
  • Detection Antibody (50X)
  • Antibody Coupled Magnetic Beads, Simplexes (50X)
  • Antibody Coupled Magnetic Beads, premixed panels (1X)
  • Competitive Conjugate Solution (50X)
  • Streptavidin-PE (SA-PE) (1X)
  • Wash Buffer Concentrate (10X)
  • Detection Antibody Diluent
  • Universal Assay Buffer (1X) and Concentrate (10X) (optional)
  • Wash Buffer PCR 8-Tube Strip
• Available Kits:
  • Simplex Kit
  • Basic Kit
  • Combinable Panels
• Required Materials Not Supplied:
  • 96-well Flat Bottom Plate

Product Usage Instructions

Assay Protocol

1. Prepare samples according to the provided instructions.
2. Add the necessary components as per the assay protocol.
3. Incubate samples with reagents for the specified time.

Instrument Settings

1. Set up the instrument following the manufacturer’s guidelines.
2. Ensure the correct parameters are selected for the assay.

Analyze Results

1. Use the appropriate software to analyze the results.
2. Interpret the data based on standard curves or controls provided.

FAQ

Q: Can the ProcartaPlexTM Human Simplex and Combinable Panels be used for diagnostic procedures?
A: No, these panels are for research use only and not intended for diagnostic procedures.

ProcartaPlex™ Human Simplex and
Combinable Panels
USER GUIDE
Publication Number  MAN0028348
Revision   B (31)

For Research Use Only. Not for use in diagnostic procedures.

Bender MedSystems GmbH | Campus Vienna Biocenter 2 | 1030 Vienna, Austria
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.

Revision history:   MAN0028348 B (31) (English)

material corrections.
A00 (30)| 18 April 2023| New document for ProcartaPlex™ Human Simplex and Combinable Panels User Guide.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
End User License Agreement for Luminex Assay Products: By opening the packaging containing this Assay Product (which contains fluorescently labeled microsphere beads authorized by Luminex Corporation) or using this Assay Product in any manner, you are consenting and agreeing to be bound by the End User Terms and Conditions and the End User License Agreement available at https://www.luminexcorp.com/end-user-terms-and-conditions/#sec3. If you do not agree to all of the terms and conditions, you must promptly return this Assay Product for a full refund prior to using it in any manner.

TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. xMAP, Luminex 100/200, MAGPIX, INTELLIFLEX, xMAP INTELLIFLEX DR-SE and FLEXMAP 3D are trademarks of Luminex Corp.
©2023-2024 Thermo Fisher Scientific Inc. All rights reserved.

Product information

Product description
The ProcartaPlex™ Human Simplex and Combinable Panels have been optimized for detection of multiple analytes from serum, plasma, and cell culture supernatants. Additionally, some assays were validated with urine, cell lysates, or cerebrospinal fluid (CSF) samples.
ProcartaPlex™ Human Simplex and Combinable Panels are designed to be combinable with each other so that you can create your own multiplex panel that utilizes Luminex™ xMAP™ technology for protein quantitation. When combining multiple Simplex Kits only one buffer kit (Basic Kit) is needed for each assay plate regardless of plex size. The buffer kits are sold separately and are optimized for the differ ent species and kit formats. When combining Simplex Kits with Combinable Panels Kits, there is no need to purchase a buffer kit. All buffers are included in the Combinable Panel Kit.
For detailed product information, visit thermofisher.com/procartaplex

Contents and storage
Upon receipt, store the kit at 2°C to 8°C. When stored as indicated, all reagents are stable until the expiration date.

Components supplied| Simplex kit| Basic kit| Combinable panels
96-well Flat Bottom Plate| | ✓| ✓
Black Microplate Lid| | ✓| ✓
Plate Seals| | ✓| ✓

1. Contains sodium azide.
2. Will be included in Competitive Assays only.

Retain the lot-specific Certificate of Analysis (CoA) that contains the product expiration date. The Certificate of Analysis also includes information required for the assay setup on the xMAP™ instrument (like bead number, analyte and S1 concentration).

Required materials not supplied
Catalog numbers that appear as links open the web pages for those products.

MLS
Vortex mixer| (Cat. No. 88882010)
Microcentrifuge| MLS
Adjustable single and multichannel pipettes with disposable tips and low volume reservoirs|

(Cat. No. 95128093)

Beakers, flasks, and cylinders necessary for preparation of reagents| MLS
Orbital microplate shaker with at least 1.5 mm or 0.059 inch orbit diameter capable of maintaining a speed of 600 ± 50 rpm| (Cat. No. 88882006)
[1] “MLS” indicates that the material is available from fisherscientific.com or another major laboratory supplier.

Note: Use of rockers or large orbit shakers can cause adverse results and are therefore not recommended to be used for ProcartaPlex™ assays.

Procedural guidelines

• Thoroughly read this user guide and the Certificate of Analysis that is included with the assay kit. A product insert may contain specific instructions for proper use of your kit.
• For FLEXMAP 3D™ and INTELLIFLEX™ instruments initiate the startup protocol to warm up the lasers for at least 30 minutes. Ensure that the Luminex™ machine is calibrated according to the manufacturer’s instructions.
• When working with samples and standards, change the pipette tips after every transfer and avoid creating bubbles when pipetting.
• During the incubation steps, cover the plate with the Black Microplate Lid provided in the kit to minimize exposure of the beads to light.
• Be careful not to invert the plate during the assay or allow contents from one well to mix with another well.
• Use a multi-channel pipette and reagent reservoirs whenever possible to achieve optimal assay precision.

Competitive assays
Competitive assays are based on competitive ELISA technique and require addition of Competitive Conjugate Solution (1X) to samples, standards, and blanks. Competitive assays available are listed below:

Note: The assay protocol for competitive assays differs from our standard assay protocol (see step 4).

Workflow

Assay protocol

| Prepare antigen standard
| Add capture beads

IMPORTANT! Simplex kits contain 50X beads. Refer to Table 4 for dilution instructions, which vary according to the number of combined Simplex Beads.

1. Vortex beads 30 sec.
2. Add 50 µL of beads to each well. Remove liquid.

Note: Wash the plate after adding the beads.
Note: If required, add next type of beads. Repeat, until all beads needed are on the plate.

| Add samples and standards

1. Add the following according to sample type:
   • For serum, plasma, urine, cell lysates, and CSF samples: Add 25 µL of Universal Assay Buffer, then add 25 µL of standards or samples. For background wells, add 50 µL of 1X UAB.
   • For cell culture supernatant samples: Add 50 µL of standards or samples. For background wells, add 50 µL of cell culture medium.
2. Seal the plate and incubate with shaking for 60–120 min at room temp.

| Add detection antibody

1. Add 25 µL of Detection Antibody Mix (1X).
2. Seal the plate and incubate with shaking for 30 min at room temp.
3. Wash plate twice.

| Add Streptavidin-PE

1. Add 50 µL of Streptavidin-PE.
2. Seal the plate and incubate with shaking for 30 min at room temp.
3. Wash plate twice.

| Resuspend beads

1. Add 120 µL of Wash Buffer.
2. Seal the plate and shake for 5 min at room temp.

| Acquire data on xMAP ™ system

Methods

Prepare the samples
Thaw frozen serum, plasma, and urine samples on ice and mix well by vortexing. Centrifuge at 10,000 × g for 5–10 minutes to pellet out particulates. Avoid multiple freeze/thaw cycles. If samples are high in lipid content, centrifuge at 10,000 × g for 10 minutes and transfer contents to a new tube.

Prepare plasma samples

1. Collect samples in sodium citrate or EDTA tubes. If using heparin as an anticoagulant, no more than 10 IU of heparin per mL of blood collected should be used to prevent assay interference that can result in a false positive signal.
2. Centrifuge samples at 1,000 × g at 4°C for 10 minutes within 30 minutes of collection.
3. Collect the plasma fraction. Use immediately or store aliquots at –80°C.

Prepare serum samples

1. Allow blood to clot for 20–30 minutes at 20–25°C.
2. Centrifuge at 1,000 × g for 10 minutes at 20–25°C.
3. Collect the serum fraction. Alternatively, a serum separator tube can be used following the manufacturer’s instructions.
4. Use immediately or store aliquots at –80°C. Avoid multiple freeze/thaw cycles.

Prepare cell culture supernatants

1. Centrifuge samples at 1,400 rpm for 10 minutes at 4°C to remove particulates.
2. Aliquot the clarified medium into clean polypropylene microcentrifuge tubes.
3. Use immediately or store aliquots at –80°C. Avoid multiple freeze/thaw cycles.

Prepare CSF samples

1. Centrifuge samples at 1,400 rpm for 10 minutes at 4°C to remove particulates.
2. Use immediately or store aliquots at –80°C. Avoid multiple freeze/thaw cycles.

Prepare urine samples

1. Centrifuge at 1,000 x g for 5–10 minutes at room temperature to remove particulates if needed.
2. Aliquot the clarified medium into clean polypropylene microcentrifuge tubes.
3. Use immediately or store aliquots at –80°C. Avoid multiple freeze/thaw cycles.

Prepare cell lysate samples
For preparing cell lysates, we recommend that you either use ProcartaPlex™ Cell Lysis Buffer (Cat. No. EPX-99999-901) or Invitrogen™ Cell Extraction Buffer (Cat. No. FNN0011) supplemented with 0.5M EDTA and Thermo Scientific™ Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (100X) (Cat. No. 78440 or 78442). For example, add 100 μL of 0.5M EDTA Solution and 100 μL of Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (100X) to 9.8 mL of Cell Extraction Buffer. Depending on your cell system other buffers can be suitable as well.

1. Stimulate cells as desired.
   a. Non-adherent cells:

   • Collect the cells by low speed centrifugation (400 × g for 10 minutes at 4℃).

   • Remove the medium from the pellet, and wash twice with ice-cold PBS by low speed centrifugation.

   • Remove the PBS, and add 0.5–1.0 × 107 cells/mL Cell Lysis Buffer yielding about 500–3000µg/mL protein according to BCA test.

   • Incubate on ice for 15 minutes, vortexing every 5 minutes.
     b. Adherent cells:

   • Remove the medium from the cells, and wash twice with ice-cold PBS.

   • Remove the PBS, and add Cell Lysis Buffer to cover the surface of the culture dish.

   • Incubate on ice for 15 minutes, vortexing every 5 minutes.
     Note: Alternatively it is possible to trypsinize, wash and lyse adherent cells as described for non-adherent cells.

2. Collect the cell lysate.

3. Transfer the lysate to a microfuge tube, then centrifuge at 13,000 rpm for 10 minutes 4℃.

4. Aliquot the cleared lysate into clean microfuge tubes. Then determine total protein concentration.

IMPORTANT!

• Proceed to analysis immediately after collection or freeze and store the cell lysates at –80°C.
• Avoid multiple freeze-thaw cycles of the frozen cell lysates. Thaw completely, mix well, then clarify by centrifugation at 18,000 × g for 5 minutes before analysis to prevent clogging.

Dilution of serum and plasma samples
You may need to further dilute your samples if the analyte concentration exceeds the assay upper limit of quantitation (ULOQ). When preparing dilution of serum and plasma samples, use Universal Assay Buffer (1X). For dilution of cell culture supernatant samples, use cell culture medium that was used to culture the cells. Recommended dilution factors for analytes with high normal serum or plasma concentration are listed in the Table 1.
Simplex kits for analytes with high concentration in serum and plasma include Universal Assay Buffer (10X).

Table 1   Human analyte dilution factors for serum and plasma samples

Analytes| Recommended sample dilution factor
Fetuin-A| 10,000
Fibrinogen| 200,000 [1]
Haptoglobin| 100
HGFR (c-Met)| 100
ICAM-1| 100
IGFBP-2| 100
IGFBP-3| 100
IL-6Ra| 100
Lactoferrin| 100
Lp-PLA2| 100
MBL| 100
MIA| 100
MIP-4 (CCL18)| 100
MIP-5 (CCL-15)| 100
MMP-2| 100
MMP-3| 100
MMP-9| 100
NGAL| 100
NRP-1| 100
Osteopontin (OPN)| 100
Periostin (OSF‑2)| 100
RANTES (CCL5)| 100
RBP4| 100
REG3a| 100
SAA| 100
SAP (Pentraxin 2)| 4,000
SCGF-β| 100
TIMP-1| 100

Analytes| Recommended sample dilution factor
VCAM-1| 100
VE-Cadherin| 100
YKL-40 (CHI3L1)| 100

Dilution of CSF samples
You may need to further dilute your samples if the analyte concentration exceeds the assay upper limit of quantitation (ULOQ). When preparing dilution of serum and plasma samples, use Universal Assay Buffer (1X). For dilution of cell culture supernatant samples, use cell culture medium that was used to culture the cells. Recommended dilution factors for analytes with high normal serum or plasma concentration are listed in Table 2.

Table 2   Human analyte dilution factors for CSF samples

Dilution of urine samples
The assays for the analytes listed in (Table 3) are validated for the use with urine samples. These targets typically have high concentrations in urine samples. We recommend that you dilute samples 1:100 in Universal Assay Buffer (1X) to ensure that they fall within range of the assay.

Table 3   Human analyte dilution factors for urine samples

Analytes| Recommended sample dilution factor
TIMP-1| 100
Uromodulin| 100

Dilution of cell lysates samples
ProcartaPlex™ Signaling Assays are validated for the use with Cell Lysates. Please refer to “Prepare Cell Lysate samples on page 10” for sample preparation. We recommend that you dilute samples 1:5 in Universal Assay Buffer (1X). Higher dilution might be necessary depending upon cell-lysates used.

Recommendations for isolation and lysis of exosomes
After isolation of exosomes by precipitation with reagents—Total Exosome Isolation Reagent (from serum) Cat. No. 4478360, (from plasma) Cat. No. 4484450, or (from cell culture media) Cat. No. 4478359—ultracentrifugation, or other procedure, lyse exosomes using Exosome Resuspension Buffer provided in the Total Exosome RNA & Protein Isolation Kit (Cat. No. 4478545) or other established procedure.
Further dilute the sample in 1X Universal Assay Buffer if needed, then immediately proceed to add samples to the plate.
Resuspension volume and predilution, if needed, depends on the exosome source, volume, and sample concentration.

TGF-β (acidification of samples)
TGF-β1 has to be acid treated for proper detection of the bioactive form. Therefore, this analyte should be tested as simplex assay. An additional datasheet with the preparation instructions are included in TGF-ß kits and can be requested from technical service.

Prepare the reagents
Before starting with the assay protocol, define the plate map. Mark the standard, sample, and background wells to determine the number of wells used (see Appendix A, “Recommended plate layout”).

Prepare 1X Wash Buffer
Bring the Wash Buffer Concentrate (10X) to room temperature and vortex for 15 seconds. Mix 20 mL of the Wash Buffer Concentrate (10X) with 180 mL ddH2O. Mix gently to avoid foaming. Wash Buffer (1X) can be stored at 2–8°C for up to 6 months.

Note: Additional Wash Buffer Concentrate (200 mL, Cat. No. EPX-66666-001) can be purchased separately for automated plate washers.

Optional: Prepare 1X Universal Assay Buffer (UAB)

IMPORTANT! This dilution step is only required for kits containing 10X Universal Assay Buffer.

Note: 1X UAB is required for the preparation of standards and dilution of serum and plasma samples, CSF and cell lysates only. If working with cell culture supernatant samples, use the cell culture medium as a diluent.

Mix 10 mL of 10X Universal Assay Buffer (UAB) with 90 mL ddH2O. Mix gently to avoid foaming. 1X UAB can be stored at 2–8°C for up to 30 days.

Prepare 1X Competitive Conjugate Solution for competitive assays
Competitive Conjugate Solution (50X) is included in Competitive Assays. Prepare the Competitive Conjugate Solution (1X) by mixing 50 μL of Competitive Conjugate Solution (50X) with 2,450 μL of Universal Assay Buffer (1X).

Prepare 1X Simplex Beads
Simplex kits are provided with concentrated 50X beads, which will require dilution before use. The dilution is dependent on the number of different Simplex Beads that are combined (Table 4).

IMPORTANT! ProcartaPlex™ Simplex Kits and/or Combinable Panels can be mixed for enhanced flexibility. Ensure that the bead regions from your ProcartaPlex™ Simplex Kits or panels do not overlap. Some analytes use the same bead region and cannot be combined in one multiplex assay. Check the compatibility of our analytes using our online panel configurator or contact our technical support.

1. Vortex each Simplex Bead vial (50X) for 30 seconds, then add 100 μL of each Simplex Bead vial (50X) to a mixing bottle if using a whole plate (otherwise adjust the volume accordingly).
2. Add Wash Buffer (1X) to a final volume of 5 mL. To combine 2 or more different Simplex Bead vials, follow Table 4 (using a whole plate):

Table 4   Dilution of Simplex Beads

Number of different Simplex Bead vials to be mixed| Total volume of mixed bead solution| Volume of Wash Buffer (1X) to add
---|---|---
1| 100 µL| 4,900 µL
2| 200 µL| 4,800 µL
3| 300 µL| 4,700 µL
4| 400 µL| 4,600 µL
5| 500 µL| 4,500 µL
6| 600 µL| 4,400 µL

Prepare 1X Detection Antibody Mixture
For simplex and combinable panels, detection antibody is provided at 50X concentration. Add 60 μL of each detection antibody concentrate to the mixing bottle and bring volume to a total of 3 mL using detection antibody diluent if using a whole plate (otherwise adjust the volume accordingly).

Number of vials of detection antibody| Total volume of detection antibody| Volume of diluent to add
---|---|---
1| 60 µL| 2,940 µL
2| 120 µL| 2,880 µL
3| 180 µL| 2,820 µL
4| 240 µL| 2,760 µL

Number of vials of detection antibody| Total volume of detection antibody| Volume of diluent to add
5| 300 µL| 2,700 µL
6| 360 µL| 2,640 µL

Prepare Standard Mix
Carefully review the Certificate of Analysis for lot-specific information on the kit components. The majority of kits are supplied with lyophilized Standard Mixes that contain a mix of multiple standard proteins. Some kits contain multiple sets of standards, each with a unique lot number, that require pooling prior to use. Each kit is shipped with two identical vials of each premixed antigen standard set from the same lot to permit the user to run the assay twice if running a partial plate. When combining multiple kits, ensure that the antigen standards of your analytes of interest are only present in one of the used standard vials. For instructions for combining more than five antigen standard sets, visit thermofisher.com/support.
When preparing antigen standards, the final volume after reconstitution and pooling should be 250 μL. For experiments measuring serum, plasma, urine, or CSF samples, use 1X UAB to reconstitute and dilute the standard. For experiments measuring cell culture supernatant samples, use fresh cell culture medium as the diluent.

Note: Change pipette tips after each dilution step and avoid air bubbles.

1. Centrifuge each different standard mix stock vial at 2,000 x g for 10 seconds.
2. Add 50 μL of diluent to each stock vial.
3. Vortex the vials at high speed for 30 seconds and centrifuge at 2,000 x g for 10 seconds to collect contents at the bottom of the vial.
4. Incubate on ice for 10 minutes to ensure complete reconstitution.
5. Pool entire content of each stock vial into one of the vials and fill up to a total volume of 250 μL.
6. Vortex the vial at high speed for 10 seconds and centrifuge at 2,000 x g for 10 seconds to collect contents at the bottom of the vials.

of standard sets| Reconstitution volume per vial| Pooled volume| Buffer to

add| Total volume
---|---|---|---|---
1| 50 μL| 50 μL| 200 μL| 250 μL
2| 50 μL| 100 μL| 150 μL| 250 μL
3| 50 μL| 150 μL| 100 μL| 250 μL
4| 50 μL| 200 μL| 50 μL| 250 μL
5| 50 μL| 250 μL| 0 μL| 250 μL

Prepare 4-fold serial dilution

1. Label the tubes in the 8-Tube Strip: Std1, Std2, Std3, Std4, Std5, Std6, and Std7.
2. Add 200 μL of the reconstituted standard mix into Std1 tube.
3. Add 150 μL of diluent into Std2–Std7 tubes.
4. Transfer 50 μL from Std1 tube into Std2 tube.
5. Mix by pipetting up and down 10 times.
6. Transfer 50 μL of the mixed standards from Std2 tube into Std3 tube using new pipette tip.
7. Mix by pipetting up and down 10 times.
8. Repeat steps 4–7 for tubes Std4–Std7, changing pipette tips between dilution steps, see Figure 1.
9. Add 150 μL of diluent to the last tube of the 8-Tube Strip to serve as a background.
10. Keep tubes on ice until ready to use.

Note: Use reconstituted standards immediately. Reconstituted standards cannot be stored. Discard unopened standard vials if the entire plate was used in a single experiment.

Assay protocol

1. Define the plate map by marking the standard, sample, and blank wells using the plate map in Appendix A, “Recommended plate layout”.
2. Add Capture Bead Mix to the plate.
   • Vortex the 1X Capture Bead Mix vial for 30 seconds at high speed.
   • Using a multichannel pipette, add 50 μL of the Capture Bead Mix to each well of the plate according to the table:
     Number of different bead vials to be mixed| Amount added to each well
     ---|---
     1| 50 µL
     2| 100 µL
     3| 150 µL
     4| 200 µL
     5| 250 µL
     6| 300 µL

IMPORTANT! If working with Simplex kits only, add 50 μL per well of diluted 1X Simplex Beads prepared according to “Prepare 1X Simplex Beads” on page 16”
Note : Combinable panels are provided with magnetic beads at a 1X working concentration. Diluted Simplex Beads (1X) can be added alone or in combination with combinable panels. Note that 50 μL per bead vial per well is required.

  * If more than 6 bead vials need to be mixed, proceed to step 3 and repeat steps 2 and 3 until all beads have been added to the plate wells and washed.

3. Wash beads using a Hand-Held Magnetic Plate Washer.
   Note: To avoid loss of beads, secure the plate using the clamps on both sides of the Hand-Held Magnetic Plate Washer during this procedure.
   Note: This protocol was developed using the Hand-Held Magnetic Plate Washer (Cat. No. EPX-55555-000). Other washers should be validated by the end user.

   • Place the plate on the Hand-Held Magnetic Plate Washer and wait 2 minutes to allow the beads to settle on the bottom of each well.
   • Remove the liquid by quickly inverting the washer/plate assembly over a sink or waste container.
   • Gently blot the inverted washer/plate assembly onto several layers of paper towels or absorbent surface to remove any residual liquid.
   • Add 150 μL of 1X Wash Buffer into each well and wait 30 seconds.
   • Remove the liquid by quickly inverting the washer/plate assembly over a sink or waste container.
   • Gently blot the inverted washer/plate assembly onto several layers of paper towels or absorbent surface to remove any residual liquid.
   • Remove the plate from the magnet and proceed to step 4.

4. Add samples and standards to the plate.

   • Serum, plasma, urine, cell lysates, and CSF: Add 25 μL of 1X UAB to each well followed by 25 μL of prepared standards or samples as defined on the plate layout. Add an additional 25 μL of 1X UAB to the wells designated as backgrounds.
   • Cell culture supernatants: Add 50 μL of prepared standards or samples as defined on the plate layout. Add 50 μL of cell culture medium to the wells designated as backgrounds.
   • For Competitive Assays only: Add 20 µL of diluted 1X Competitive Conjugate Solution to each well and proceed to substep 4d. If not working with a Competitive Assay, skip this step and proceed to substep 4d.
   • Seal the plate using one of the provided Plate Seals and cover with the provided Microplate Lid. Shake at 600 rpm for 60–120 minutes at room temperature.
     IMPORTANT! If only working with Competitive Assays, proceed directly to step 7.

5. Remove and discard the Plate Seal. Wash the plate following the steps below.

   • Place the plate on the Hand-Held Magnetic Plate Washer and wait 2 minutes to allow particles to settle on the bottom of each well.
   • Remove the liquid by quickly inverting the washer/plate assembly over a sink or waste container.
   • Gently blot the inverted washer/plate assembly onto several layers of paper towels or absorbent surface to remove any residual liquid.
   • Add 150 μL of 1X Wash Buffer into each well and wait 30 seconds.
   • Remove the liquid by quickly inverting the washer/plate assembly over a sink or waste container.
   • Gently blot the inverted washer/plate assembly onto several layers of paper towels or absorbent surface to remove any residual liquid.
   • Repeat steps 5d-f once for a total of two washes.
   • Remove the plate from the magnet and proceed to step 6.

6. Add Biotinylated Detection Antibody Mix to the plate.

   • Using a multichannel pipette, add 25 μL of the detection antibody solution to each well of the plate. Gently tap the plate to evenly distribute the solution in the wells.
   • Seal the plate using a new Plate Seal and cover with the provided Microplate Lid. Shake at 600 rpm for 30 minutes at room temperature.

7. Wash the plate following step 5.

8. Add Streptavidin-PE (SA-PE) to the plate.

   • Add 50 μL of SA-PE solution to each well.
   • Seal the plate using new Plate Seal and cover with the provided Microplate Lid. Shake at 600 rpm for 30 minutes at room temperature.

9. Wash the plate following step 5.

10. Prepare the plate for immediate or later analysis on a xMAP™ instrument.

    • Add 120 μL of Wash Buffer (1x) into each well.
    • Seal the plate using new Plate Seal and cover with the provided Microplate Lid. Shake at 600 rpm for 5 minutes at room temperature.
      Note : Alternatively, it is also possible to store the 96-well plate overnight at 4°C. On the next day, shake the plate at 600 rpm for 5 minutes at room temperature and proceed with the next step below.

11. Remove the Plate Seal and run the plate on a xMAP™ instrument.

Instrument settings

Follow the recommended guidelines and procedures for calibration and verification of the instrument. Laser-based systems require 30 minutes to warm up prior to use.

Instrument

| Acquisition volume| Timeout (optional)|

Bead type

|

DD gate

|

Reporter gain

| Min. bead count
---|---|---|---|---|---|---
MAGPIX™| 50 μL[1]| NA| MagPlex™| NA| Standard PMT| 50
xMAP™ INTELLIFLEX™ DR-SE

xMAP™ INTELLIFLEX™

| 30 μL| 40 seconds| MagPlex™| 7,000–

17,000

| Standard PMT| 50
FLEXMAP 3D™

Luminex™ 100/200™

| 50 μL| 60 seconds| MagPlex™| 7,500–

25,000

| Standard PMT| 50
Bio-Rad™ Bio-Plex™| 50 μL| 60 seconds| MagPlex™| 5,000–

25,000

| Standard PMT| 50
[1] MAGPIX volume can be changed during the run to optimize bead count.

Note: To assure a good bead count, the probe height must be adjusted to the plate provided in the kit. We recommend using two 5.08 mm spacer disks to adjust the sample probe height for Mylar-bottom plates.

Analyze results
The concentration of the samples can be calculated by plotting the expected concentration of the standards against the NET MFI generated by each standard. For Bio-Plex™ Manager, plot standard concentrations against FI-Bkgd. A 4PL or 5PL algorithm is recommended for the best curve fit. Analyze the assayed samples according to the operation manual for the Luminex™ or Bio-Plex™ instrument.
We offer a free and robust analysis software package for data analysis. To analyze the data, follow the instructions below or contact our technical support.

1. Export the run data in .csv format and navigate to the ProcartaPlex™ Analysis App on Thermo Fisher Connect: https://apps.thermofisher.com/apps/procartaplex
   Note : Before exporting .csv raw data from Bio-Plex™ Manager, please make sure to set ‘Analytes Labels’ under ‘Document Export Properties’ to ‘Name (Region)’. The .csv raw data exported as Report Type ‘xPONENT’ from INTELLIFLEXTM instruments are supported.

2. Upload the .csv files to the ProcartaPlex™ Analysis App to analyze the run data. The intuitive software features 4PL/5PL curve fit optimization, group-wise statistical and heat map analysis. Users can export detailed reports including images for presentations and publications.

Note: The sample dilution factor must be accounted for in the software analysis.

IMPORTANT! For ProcartaPlex™ getting started guides, technical literature, protocol support tools, and common troubleshooting questions visit thermofisher.com/procartaplexsupport For more complete troubleshooting questions and answers, visit our FAQ database at thermofisher.com/procartaplexfaqs

A Recommended plate layout

B Documentation and support

Customer and technical support

Visit thermofisher.com/support for the latest service and support information.

• Worldwide contact telephone numbers
• Product support information
  • Product FAQs
  • Software, patches, and updates
  • Training for many applications and instruments
• Order and web support
• Product documentation
  • User guides, manuals, and protocols
  • Certificates of Analysis
  • Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies’ General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and- conditions.html. If you have any questions, please contact Life Technologies at www.thermofisher.com/support.

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References

• Laboratory & Production Essentials | Fisher Scientific
• Thermo Fisher Scientific - US
• Search Results Page
• Help and Support | Thermo Fisher Scientific - AU
• Symbols | Thermo Fisher Scientific - UK
• Help and Support | Thermo Fisher Scientific - AU
• Thermo Fisher Scientific - US
• ProcartaPlex
• Laboratory & Production Essentials | Fisher Scientific
• Laboratory & Production Essentials | Fisher Scientific
• Total Exosome Isolation Reagent (from cell culture media)
• Total Exosome Isolation Reagent (from serum)
• Total Exosome RNA & Protein Isolation Kit
• Total Exosome Isolation Kit (from plasma)
• Halt™ Protease and Phosphatase Inhibitor Cocktail (100X)
• Digital Microplate Shakers
• Digital Vortex Mixers
• Reagent Reservoirs
• Hand-Held Magnetic Plate Washer
• ProcartaPlex™ Wash Buffer (10X)
• EPX-99999-901
• Cell Extraction Buffer
• ProcartaPlex Panel Configurator
• Search Results Page
• Symbols | Thermo Fisher Scientific - US
• Help and Support | Thermo Fisher Scientific - US

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