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HUMAN CYTOKINE/CHEMOKINE PREMIXED

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 Luminex¹⁰⁰.

I. INTENDED USE

This is a multiplex assay kit manufactured by Linco Research to be used for the simultaneous quantitative determination of the following human cytokines and chemokines: IL-1α, IL-1b , IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, Eotaxin, IFNg, G-CSF, GM-CSF, IP-10, MCP-1, MIP-1α, RANTES and TNFa premixed as a 13, 21, or 22 plex. This kit may be used for the analysis of all of the above cytokines and chemokines in tissue/cell lysate and culture supernatant samples. This kit may also be used in undiluted serum, plasma, other body fluids, and tissue extract samples for the analysis of all of the above cytokines and chemokines except RANTES because of high concentrations of RANTES in the blood. Serum or plasma samples should be diluted 1:50 when the kit is used for analyzing RANTES. Single-plex RANTES, IL-10, and TNFα kits are also available from Linco Research

This kit is for research purposes only.

II. REAGENTS SUPPLIED

A. Antibody-Immobilized Premixed Beads

Quantity: 3.5ml/bottle

The following bead sets are premixed in the Bead Bottle:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

B. Human Cytokine/Chemokine Standard Cocktail

1 vial containing human cytokine standard cocktail, lyophilized

Quantity: 1 vial

C. Human Cytokine/Chemokine Quality Controls

Control I – 1 vial containing mixed cytokine cocktail, lyophilized

Control II – 1 vial containing mixed cytokine cocktail, lyophilized

Quantity: 1 vial/Control

D. Serum Matrix, lyophilized (optional – for serum/plasma samples)

Serum containing 0.08% Sodium Azide

Quantity: 1 ml/vial

E. Human Cytokine/Chemokine Detection Antibodies

1 bottle containing a cocktail of biotinylated detection antibodies in Assay Buffer

Quantity: 3.2 ml/bottle

F. Streptavidin-Phycoerythrin

1 bottle containing Streptavidin-Phycoerythrin prepared in Assay Buffer

Quantity: 3.2 ml/bottle

G. Assay Buffer

50 mM PBS with 25 mM EDTA, 0.08% Sodium Azide, 0.05% Tween 20, and 1% BSA, pH 7.4.

Quantity: 30 ml/bottle

H. 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

I. Microtiter Filter Plate

Quantity: 1- 96 Well Filtration Plate

J. 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, immediately 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.

IV. REAGENT PRECAUTIONS

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. On disposal, 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 Pipettes with Tips capable of delivering 25 m l to 1000 m l

2. Multichannel Pipettes capable of delivering 5 m l to 50 m l or 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, Model #4625, or equivalent)

10. Vacuum Filtration Unit (Millipore Vacuum Manifold Catalogue #MAVM0960R, or equivalent)

11. Luminex¹⁰⁰ by Luminex Corporation

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 debris and assay immediately or aliquot and store samples at £ -20ºC. Avoid multiple (>2) freeze/thaw cycles. Tissue Culture Supernatant may require a dilution with the appropriate medium prior to assay.

C. Preparation of Serum 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. Except for RANTES, use Serum Matrix as the diluent for quantifying cytokines/chemokines in serum or plasma samples. When RANTES is quantified, Assay Buffer should be used as diluent for blood samples. When RANTES is measured, samples should be diluted 1:50 in the assay buffer and a standard curve with assay buffer matrix should be used accordingly.

D. Preparation of Plasma Samples:

Plasma collection using EDTA as an anti-coagulant is recommended. Centrifuge for 10 minutes at 1000 Xg within 30 minutes of blood collection. Remove plasma and assay immediately or aliquot and store samples at £ -20ºC. Avoid multiple (>2) freeze/thaw cycles. Except for RANTES, use Serum Matrix as the diluent for quantifying cytokines/chemokines in serum or plasma samples. When RANTES is quantified, Assay Buffer should be used as diluent for blood samples. When RANTES is measured, samples should be diluted 1:50 in the assay buffer and a standard curve with assay buffer matrix should be used accordingly.

E. All samples must be stored in polypropylene tubes. DO NOT STORE SAMPLES IN GLASS.

F. Avoid using samples with gross hemolysis or lipemia.

G. Care must be taken when using heparin as an anticoagulant, since an excess of heparin will provide falsely high values. Use no more than 10 IU heparin per ml of blood collected.

VII. TECHNICAL GUIDELINES

To obtain reliable and reproducible results, the operator should carefully read this entire manual and fully understand all aspects of each assay step before running 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 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. If, however, the plate cannot be read immediately, seal the plate, cover with aluminum foil, and store at 2-8° C for up to 24 hours. Prior to reading, agitate the plate on the plate shaker at room temperature for 10 minutes. Delay in reading a plate may result in decreased sensitivity for some cytokines and chemokines.

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

Antibody-Immobilized Beads are premixed. Sonicate and/or vortex before use.

B. Preparation of Human Cytokine Standard Cocktail

1.) Prior to use, reconstitute the Human 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 appropriately labeled polypropylene microfuge tube. This will be used as the 10,000 pg/ml standard; the unused portions may be stored at £ -20° 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.