A Multiplex Bead-Based Immunoassay to Quantify Multiple Cytokine Targets

1. Biological Sample Preparation

NOTE: The chosen anatomical site and volume of blood collection are left to the discretion of each individual investigator and will not affect the outcome of the assay. However, once they are obtained, samples should be handled according to the steps listed below.

1. Preparation of Serum Samples
   1. Collect the desired volume of blood into the preferred collection tube and allow it to clot for at least 30 min.
   2. Centrifuge the sample for 10 min at 1,000 x g at room temperature.
   3. Remove serum and assay immediately or aliquot into polypropylene microcentrifuge tubes and store samples at ≤ -20 °C.
2. Preparation of Plasma Samples
   1. Collect the desired volume of blood using ethylenediaminetetraacetic acid (EDTA) coated blood collection tubes.
   2. Centrifuge for 10 min at 1,000 x g at room temperature within 30 min of collection.
   3. Remove plasma and assay immediately, or aliquot into polypropylene microcentrifuge tubes and store samples at ≤ -20 °C.
3. Preparation of Tissue Culture Supernatant Samples
   1. Centrifuge the sample for 10 min at 1,500 x g at 4 °C to remove cells and debris.
   2. Collect supernatant and assay immediately or aliquot into polypropylene microcentrifuge tubes and store at ≤ -20 °C.
4. Thawing of Frozen Biological Samples
   1. Completely thaw any previously frozen biological samples on ice and vortex briefly before use. Avoid more than 2 freeze/thaw cycles.
      NOTE: The samples used in this protocol were obtained by culturing BALB/c mouse splenocytes (1 x 10⁶ cells at 37 °C + 5% CO₂ under various conditions: unstimulated, LPS (100 ng/mL), αCD3 (1 µg/mL plate-coated) + αCD28 (1 µg/mL soluble), PMA (20 ng/mL) + Ionomycin (500 ng/mL). Culture supernatants were collected after 48 h and processed as described in section 1.3.

2. Reagent Preparation

1. Preparation of Pre-mixed Antibody-Immobilized Beads
   1. Sonicate pre-mixed beads bottle for 1 min in a sonicator bath at room temperature, and then vortex for 30 s prior to use. If no sonicator bath is available, increase the vortex time to 1 min.
2. Preparation of Wash Buffer
   1. Bring the 20x Wash Buffer (20x PBS with 1% Tween-20) supplied with the kit to room temperature and vortex to bring all salts into the solution.
   2. Dilute 25 mL of 20x Wash Buffer with 475 mL of deionized water. Store unused portions between 2 °C and 8 °C for up to one month.
3. Preparation of Matrix B (For Use with Serum and Plasma Samples Only)
   1. Add 5.0 mL of the Assay Buffer (PBS with 1% BSA) included in the kit to the bottle containing lyophilized Matrix B. Allow at least 15 min for complete reconstitution, then vortex to mix well. Leftover reconstituted Matrix B can be stored at ≤ -70 °C for up to one month.

3. Standard Preparation

NOTE: Each analyte in this panel has a top standard concentration of 10,000 pg/mL.

1. Add 250 µL of Assay Buffer to reconstitute the lyophilized Mouse Th Cytokine Standard Cocktail. Mix by briefly vortexing and allow the vial to sit at room temperature for 10 min.
2. Transfer the standard cocktail to a polypropylene microcentrifuge tube labeled "C7". This will be used as the top standard.
3. Label 6 polypropylene microcentrifuge tubes as C6, C5, C4, C3, C2, and C1.
4. Add 75 µL of Assay Buffer to each of these tubes.
5. Transfer 25 µL of the top standard C7 to the C6 tube and mix well by vortexing. This will be the C6 standard.
6. Continue performing serial 1:4 dilutions by using a new pipet tip for each tube to add 25 µL of the previous standard to the 75 µL of Assay Buffer in the next lowest standard tube, followed by vortexing to obtain standards C5, C4, C3, C2, and C1. Use Assay Buffer as the 0 pg/mL standard (C0).

4. Sample Dilution

NOTE: Preliminary pilot experiments using this assay with multiple dilutions may be required to determine the most appropriate dilution factor for a particular set of biological samples. A proper dilution factor will produce concentration calculations for the sample that lie within the bounds of the standard curve. The following steps are meant as guidelines and may have to be determined empirically depending on the sample type.

1. Dilute serum or plasma samples 2-fold with Assay Buffer (e.g. dilute 50 µL of sample with 50 µL of Assay Buffer) in polypropylene microcentrifuge tubes.
   NOTE: If further sample dilution is required, dilutions should be done with Matrix B instead of assay buffer to ensure accurate measurement. Adding serum or plasma samples without dilution will result in low assay accuracy and may clog the filter plate. Matrix B is comprised of pooled mouse serum depleted of endogenous assay targets. It is used as a serum or plasma sample diluent to avoid matrix effects, which are known to affect the analytical sensitivity of immunoassays.
2. Test cell culture supernatant samples without dilution.
   NOTE: The levels of analyte can vary greatly from sample to sample. If necessary, dilute supernatant samples using a fresh preparation of their corresponding cell culture medium or Assay Buffer.

5. Assay Procedure

NOTE: The assay can be performed in polypropylene filter plates, micro fluorescence-activated cell sorting (FACS) tubes, or V-bottom microplates. The filter plate assay procedure is recommended due to good sample-to-sample consistency, assay robustness, and ease of handling. This procedure requires a vacuum filtration unit for washing (provided by the end user).

1. Allow all reagents to warm to room temperature (20 – 25 °C) before use.
2. Set the filter plate on an inverted plate cover at all times during the assay setup and incubation steps so that the bottom of the plate does not touch any surfaces, as it may cause leaking.
3. Keep the plate upright during the entire assay procedure, including the washing steps, to avoid losing beads.
4. Keep the plate in the dark or wrapped with aluminum foil for all incubation steps.
5. Run all standards and samples as duplicates arranged on the plate in a sequential order.
6. Pre-wet the filter plate by adding 100 µL of 1x Wash Buffer to each well and let it sit for 1 min at room temperature (if using filter-bottom microplate).
7. Remove buffer volume by using the vacuum manifold (5 – 10 s). Do not exceed 10 " Hg of vacuum. Blot excess Wash Buffer from the bottom of the plate by pressing the plate on a stack of clean paper towels. Place the plate on top of the inverted plate cover.
   NOTE: Steps 5.1 – 5.2 may be omitted if the assay is performed using a V-bottom microplate or micro FACS tubes.
8. For cell culture supernatant samples, add 25 µL of Assay Buffer to all wells. Add 25 µL of each standard to the standard wells. Add 25 µL of each sample to the sample wells.
9. For measuring serum or plasma samples, add 25 µL of Matrix B to the standard wells. Add 25 µL of Assay Buffer to the sample wells. Add 25 µL of each standard to the standard wells. Add 25 µL of each diluted serum or plasma sample to the sample wells.
10. Vortex mixed beads for 30 s. Add 25 µL of mixed beads to each well, shaking the bead bottle intermittently to avoid bead settling. The final volume should be 75 µL in each well after the addition of beads.
11. Seal the plate with a plate sealer. Wrap the entire plate, including the inverted plate cover, with aluminum foil. Place the plate on a plate shaker, secure it, and shake at approximately 500 rpm for 2 h at room temperature. To prevent leaking, do not apply positive pressure to the plate sealer.
12. Without inverting, place the plate on the vacuum manifold apply vacuum as before and add 200 µL of 1x wash buffer to each well.
13. Remove the contents of the assay plate wells by vacuum filtration. Blot excess wash buffer from the bottom of the plate with an absorbent pad or paper towels. Repeat this step one more time.
    NOTE: If the assay is performed using a V-bottom microplate or micro FACS tubes skip steps 1.12 and 1.13. Instead centrifuge the plate at 1,000 x g for 5 min at room temperature, then remove the supernatant using a multichannel pipette.
14. Add 25 µL of detection antibodies (see the Table of Materials) to each well.
15. Seal the plate with a fresh plate sealer. Wrap the entire plate, including the inverted plate cover, with aluminum foil.
16. Place the plate on a plate shaker and shake at approximately 500 rpm for 1 h at room temperature.
17. Without vacuuming, add 25 µL of streptavidin-phycoerythrin (SA-PE) reagent directly to each well. No dilution of the reagent is necessary.
18. Seal the plate with a fresh plate sealer. Wrap the entire plate, including the inverted plate cover, with aluminum foil.
19. Place the plate on a plate shaker and shake at approximately 500 rpm for 30 min at room temperature.
20. Repeat step 1.13 above.
21. Add 200 µL of 1x Wash Buffer to each well. Re-suspend the beads on a plate shaker for 1 min.
22. Using a multichannel pipet, transfer samples from the filter plate to FACS tubes to read samples on a flow cytometer.
    NOTE: Sample volume may be increased from 200 µL to 300 µL by adding an extra 100 µL of 1x Wash Buffer to each tube to avoid the sample running dry. If necessary, samples may be stored at 4 °C, protected from light, and analyzed the following day. However, prolonged sample storage can lead to reduced signal.