MOUSE CYTOKINE/CHEMOKINE LINCOplex
By Purchasing this product, which contains fluorescently labeled microsphere beads authorized by Luminex Corporation ("Luminex"), you, the customer, acquire the right under Luminex’s patent rights, if any, to use this product or any portion of this product, including without limitation the microsphere beads contained herein, only with Luminex’s laser based fluorescent analytical test instrumentation marketed under the name of Luminex*.
*Luminex instrumentation refers to Luminex®100, Luminex 200 and other Luminex instruments from MiraiBio® (MasterPlex CT™), ACS® (STarStation™), Bio-Rad Laboratories, Inc.® (Bio-Plex™) and Qiagen® (LiquiChip™). For technical assistance on running LINCOplex Kits, contact our technical service department Toll Free U.S. at 866-441-8400 or 636-441-8400 or email us at info@lincoresearch.com.
This multiplex assay kit manufactured by Linco Research is to be used for the simultaneous quantification of the following mouse cytokines and chemokines in any combination: mIL-1α, mIL-1b , mIL-2, mIL-4, mIL-5, mIL-6, mIL-7, mIL-9, mIL-10, mIL-12(p70), mIL-13, mIL-15, mIL-17, mIFNg, mIP-10, mG-CSF, mGMCSF, mTNFa , mKC, mMCP-1, mMIP-1α, and mRANTES. This kit may be used for the analysis of the above cytokines and chemokines in mouse serum, plasma, tissue extract, or cell/tissue culture samples.
This kit is for research purposes only.
A. Antibody-Immobilized Beads
The following beads sets are available for your customized orders:
#26-Mouse IL-1α
#21-Mouse IL-1b
#36-Mouse IL-2
#52-Mouse IL-4
#56-Mouse IL-5
#58-Mouse IL-6
#59-Mouse IL-7
#66-Mouse IL-9
#60-Mouse IL-10
#62-Mouse IL-12(p70)
#68-Mouse IL-13
#69-Mouse IL-15
#70-Mouse IL-17
#19-Mouse IFNg
#32-Mouse IP-10
#30-Mouse G-CSF
#08-Mouse GMCSF
#64-Mouse TNFa
#04-Mouse MIP-1a
#13-Mouse MCP-1
#15-Mouse KC
#17-Mouse RANTES
Mix beads and dilute with Assay Buffer as described in Section VIII. D.
Quantity: 200μl antibody-immobilized beads per bottle
B. Mouse Cytokine/Chemokine Standard Cocktail
1 vial containing mouse cytokine/chemokine standard cocktail, lyophilized
Quantity: 1 vial
C. Mouse Cytokine/Chemokine Quality Controls
Control I – 1 vial containing mixed mouse cytokine/chemokines, lyophilized
Control II – 1 vial containing mixed mouse cytokine/chemokines, lyophilized
Quantity: 1 vial each control
D. Mouse Cytokine/Chemokine Detection Antibodies
1 bottle containing a cocktail of biotinylated detection antibodies in Assay Buffer
Quantity: 3.2 ml/bottle
E. Streptavidin-Phycoerythrin
1 bottle containing Streptavidin-Phycoerythrin in Assay Buffer
Quantity: 3.2 ml/bottle
F. Assay Buffer
PBS with 0.08% Sodium Azide and 1% BSA, pH 7.6
Quantity: 30 ml/bottle
G. 10X Wash Buffer
1:10 dilution required with deionized water to give 10 mM PBS with 0.05% Proclin, and 0.05% Tween 20, pH 7.4.
Quantity: 30 ml/bottle
H. Serum Matrix , Lyophilized
Required for serum or plasma samples only.
Serum containing 0.08% Sodium Azide
Reconstitute with 1.0 mL of deionized water before use.
Quantity: 1 ml/vial
I. Mixing Bottle
Quantity: Bottle
J. Microtiter Filter Plate
Quantity: 1- 96-Well Filtration Plate
K. Plate Sealers
Quantity: 2 Plate Sealers
III. STORAGE CONDITIONS UPON RECEIPT
Recommended storage for kit components is 2 - 8°C. See individual vials for long-term storage recommendations.
Once the standards and controls have been reconstituted, transfer contents into polypropylene vials. DO NOT STORE RECONSTITUTED STANDARDS OR CONTROLS IN GLASS VIALS. For long-term storage, freeze reconstituted standards and controls at £ -20°C. Avoid multiple (>2) freeze/thaw cycles.
DO NOT FREEZE Antibody-Immobilized Beads, Detection Antibody, and Streptavidin-Phycoerythrin.
Sodium azide has been added to some reagents as a preservative. Although the concentrations are low, sodium azide may react with lead and copper plumbing to form highly explosive metal azides. Flush with a large volume of water to prevent azide build-up.
V. MATERIALS REQUIRED BUT NOT PROVIDED
A. Reagents
Luminex Sheath Fluid (Luminex Catalogue #40-50000)
B. Instrumentation/Materials
1. Adjustable Pipettors with Tips capable of delivering 25 m l to 1000 m l
2. Multichannel Pipettor capable of delivering 25 m l to 200 m l
3. Reagent Reservoirs
4. Polypropylene Microfuge Tubes
5. Aluminum Foil
6. Absorbent Pads
7. Laboratory Vortex
8. Sonicator (Branson Ultrasonic Cleaner, Model # B200 or equivalent)
9. Titer Plate Shaker (Lab-Line Instruments, Inc. Model 4625, or equivalent)
10. Vacuum Filtration Unit (Millipore Vacuum Manifold Catalogue #MAVM0960R, or equivalent)
11. Luminex Instrument
VI. SPECIMEN COLLECTION AND STORAGE
A. A maximum of 25 m l per well of serum or plasma can be used. Tissue culture or other media may also be used.
B. Preparation of Tissue Culture Supernatant:
Centrifuge the sample to remove cell debris and run the assays immediately or aliquot and store samples at £ -20ºC. Avoid multiple (>2) freeze/thaw cycles. Tissue culture supernatant may require a dilution with the appropriate control medium prior to assay.
C. Preparation of Mouse Serum or Plasma Samples:
Allow the blood to clot for at least 30 minutes before centrifugation for 10 minutes at 1000 xg. Remove serum and assay immediately or aliquot and store samples at £ -20ºC. Avoid multiple (>2) freeze/thaw cycles. It is recommended to centrifuge samples again prior to assay setup. If serum or plasma samples need to be diluted, the Serum Matrix should be used as the sample diluent. Additional Serum Matrix is available from LINCO Research Inc.
D. All samples must be stored in polypropylene tubes. DO NOT STORE SAMPLES IN GLASS.
To obtain reliable and reproducible results, the operator should carefully read this entire manual and fully understand all aspects of each assay step before attempting to run the assay. The following notes should be reviewed and understood before the assay is set-up.
A. The Antibody-Immobilized Beads are light sensitive and must be covered with aluminum foil at all times. Cover the assay plate containing beads with aluminum foil during all incubation steps.
B. It is critical to allow all reagents to warm to room temperature (20-25° C) before use in the assay.
C. The bottom of the Microtiter Filter Plate should not be in direct contact with any absorbent material during assay set-up or incubation times. The plate can be set on the non-flat side of the plate cover or any other plate holder to raise the plate from the surface.
D. After the wash steps, keep the bottom of the Microtiter Filter Plate clean by blotting on paper towels or absorbent pads to prevent any leakage during assay set-up and incubation steps due to capillary action.
E. Keep the vacuum suction on the plate as low as possible. It is recommended to have a vacuum setting that will remove 200 ml of buffer in > 5 seconds (equivalent to < 100 mmHg).
F. After hydration, all standards and controls must be transferred to polypropylene tubes.
G. The standards prepared by serial dilution must be used within 1 hour of preparation. Discard any unused standards except the 10 ng/ml stock standard which may be stored at £ -20° C for 1 month and at £ -80° C for greater than one month.
H. Any unused mixed Antibody-Immobilized Beads may be stored in the bead mix bottle at 2-8°C for up to one month.
I. During the preparation of the standard curve, make certain to vortex the higher concentration well before making the next dilution. Use a fresh tip with each dilution.
J. The plate should be read immediately after the assay is finished. Delay in reading the plate may result in decreased signals and sensitivities for some cytokines.
K. The titer plate shaker should be set at a speed to provide maximum agitation without splashing of liquid outside the wells. For the recommended plate shaker, this would be a setting of 5-7 which is approximately 500-800 rpm.
L. Ensure the needle probe is clean. This may be achieved by sonication and/or Alcohol Flushes. Adjust probe height to the Lincoplex filter plate prior to reading an assay.
M. For cell culture supernatants or tissue extraction, use the culture or extraction medium as matrix in blank, standard curve and controls. If samples are diluted in Assay Buffer, use the Assay Buffer as matrix.
VIII. PREPARATION OF REAGENTS FOR IMMUNOASSAY
A. Preparation of Antibody-Immobilized Beads
Sonicate each antibody-bead bottle for 30 seconds; vortex for 1 minute. Add 150 µl from each antibody bead bottle to the Mixing Bottle and bring final volume to 3.0ml with Assay Buffer. Vortex well. Unused portions may be stored at 2-8°C for up to one month.
Example 1: When using 16 cytokine/chemokines antibody-immobilized beads, add 150 µl from each of the sixteen bead sets to the mixing bottle. Add 0.6 ml Assay Buffer.
Example 2: When using 8 cytokine/chemokines antibody-immobilized beads, add 150 µl from each of the eight bead sets to the mixing bottle. Add 1.8 ml Assay Buffer.
Example 3: When using 20, 21 or 22 cytokine/chemokines antibody-immobilized beads, add 150 µl from each of the 20, 21 or 22 bead sets to the mixing bottle. No Assay Buffer is added. The final volume is 3.0, 3.15, or 3.3ml, respectively.
B. Preparation of Mouse Cytokine Standard Cocktail
1.) Before use, reconstitute the Mouse Cytokine Standard Cocktail with 250 ml Deionized Water to give a 10,000 pg/ml concentration of standard. Invert the vial several times to mix. Vortex the vial for 10 seconds. Allow the vial to set for 5-10 minutes, and then transfer the standard to an appropriately labeled polypropylene microfuge tube. This will be used as the 10,000 pg/ml standard; the un-used portions may be stored at £ -20° C for up to one month.
2). Preparation of Working Standards
Label five polypropylene microfuge tubes 2000, 400, 80, 16, and 3.2 pg/ml. Add 200 ml of Assay Buffer to each of the five tubes. Prepare serial dilutions by adding 50 ml of the 10,000 pg/ml reconstituted standard to the 2000 pg/ml tube, mix well and transfer 50 ml of the 2000 standard to the 400 pg/ml tube, mix well and transfer 50 ml of the 400 standard to the 80 pg/ml tube, mix well and transfer 50 ml of the 80 standard to 16 pg/ml tube, mix well and transfer 50 ml of the 16 pg/ml standard to the 3.2 pg/ml tube and mix well. The 0 pg/ml standard (Background) will be Assay Buffer.