MOUSE CYTOKINE LINCOplex KIT ONE PLEX TNF-a

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 [email protected]

 

I. INTENDED USE

This multiplex assay kit manufactured by Linco Research is to be used for the simultaneous quantification of the following mouse cytokines and chemokines: 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.

II. REAGENTS SUPPLIED

A. Antibody-Immobilized Beads

Quantity: 3.5 ml/bottle

The following beads sets are premixed in the Bead bottle:

10 Plex Premix Beads   16 Plex Premix Beads   22 Plex Premix Beads
#21-Mouse IL-1b   #21-Mouse IL-1b   #21-Mouse IL-1b
#36-Mouse IL-2   #36-Mouse IL-2   #36-Mouse IL-2
#52-Mouse IL-4   #52-Mouse IL-4   #52-Mouse IL-4
#56-Mouse IL-5   #56-Mouse IL-5   #56-Mouse IL-5
#58-Mouse IL-6   #58-Mouse IL-6   #58-Mouse IL-6
#60-Mouse IL-10   #60-Mouse IL-10   #60-Mouse IL-10
#62-Mouse IL-12(p70)   #62-Mouse IL-12(p70)   #62-Mouse IL-12(p70)
#64-Mouse TNFa   #64-Mouse TNFa   #64-Mouse TNFa
#19-Mouse IFNg   #19-Mouse IFNg   #19-Mouse IFNg
#08-Mouse GMCSF   #08-Mouse GMCSF   #08-Mouse GMCSF
    #04-Mouse MIP-1a   #04-Mouse MIP-1a
    #13-Mouse MCP-1   #13-Mouse MCP-1
    #15-Mouse KC   #15-Mouse KC
    #17-Mouse RANTES   #17-Mouse RANTES
    #66-Mouse IL-9   #66-Mouse IL-9
    #68-Mouse IL-13   #68-Mouse IL-13
        #26-Mouse IL-1α
        #59-Mouse IL-7
        #69-Mouse IL-15
        #70-Mouse IL-17
        #32-Mouse IP-10
        #30-Mouse G-CSF

B. Mouse Cytokine/Chemokine Standard Cocktail

1 vial containing mouse cytokine/chemokine standard cocktail, lyophilized

Quantity: 1 vial

C. Mouse Cytokine/Chemokine 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: 27 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. 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, 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. 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 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.

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

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 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).

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. 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.

I. 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.

J. The titre 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.

K. 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 toe reading an assay.

L. 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 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° 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.

Standard Concentration
(pg/ml)

Volume of Deionized Water to Add

Volume of Standard
to Add

10,000

250 ml

0

Standard Concentration
(pg/ml)

Volume of Assay Buffer to Add

Volume of Standard
to Add

2000

200 ml

50 µl of 10,000 pg/ml

400

200 ml

50 µl of 2000 pg/ml

80

200 ml

50 µl of 400 pg/ml

16

200 ml

50 µl of 80 pg/ml

3.2

200 ml

50 µl of 16 pg/ml

C. Preparation of Controls

Before use, reconstitute each Mouse Cytokine Control I and Mouse Cytokine Control II with 250 ml deionized water. Invert the vials several times to mix and vortex. Allow the vials to set for 5-10 minutes and then transfer the controls to appropriately labeled polypropylene microfuge tubes. Unused portions may be stored at £ -20° for up to one month.

D. Preparation of Wash Buffer

Bring the 10X Wash Buffer to room temperature and mix to bring all salts into solution. Dilute 30 ml of 10X Wash Buffer with 270 ml deionized water. Store unused portions at 2-8° C for up to one month.

E. Preparation of Serum Matrix

This step is required for serum or plasma samples only.

Add 1.0 mL of deionized water to the vial containing the lyophilized Serum Matrix, gently swirl the bottle and then place the bottle on bench for 5 to 10 min.

IX. IMMUNOASSAY PROCEDURE

Prior to beginning this assay, it is imperative to read this protocol completely and to thoroughly understand the Technical Guidelines outlined in Section VII.

Allow all reagents to warm to room temperature (20-25° C) before use in the assay.

1. Diagram placement of Standards, 0 (Background), 3.2, 16, 80, 400, 2000, and 10,000 pg/ml, Controls I and II, and samples on Well Map Worksheet in a vertical configuration. (Note: the instrument will only read the 96-well plate vertically). It is recommended to run the assay in duplicate.

2. Block the filter plate by pipetting 200 µL of Assay Buffer into each well of the microtiter plate. Seal and mix on a plate shaker for 10 minutes at room temperature (20-25° C).

3. Remove Assay Buffer by vacuum. (NOTE: DO NOT INVERT PLATE). Remove any excess Assay Buffer from the bottom of the plate by blotting on an absorbent pad or paper towels.

4. Add 25 µL of Assay Buffer to the 0 Standard (Background).

5. Add 25 µL of Assay Buffer to the Sample wells.

6. Add 25 µL of each Standard or Control into the appropriate wells.

7. Add 25 µL of an appropriate matrix solution to the Background, Standards, and Control wells. When assaying serum or plasma samples, use the Serum Matrix provided in the kit as the matrix solution and sample diluent. When assaying non-blood samples (tissue or cell culture medium, tissue extracts), use the same matrix as the samples for the Background, Standards, and Control wells and as the sample diluent. (For example, use control tissue culture medium for tissue culture samples; use extract buffer for tissue extract samples.)

8. Add 25 µL of sample into the appropriate wells.

9. Vortex and/or sonicate Bead Bottle for approximately 30 – 60 seconds and add 25 µL of Mixed Beads to each well. (Note: during addition of Mixed Beads, shake bead mix intermittently to avoid settling.)

10. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker for 2 hours at room temperature (20-25° C) or overnight (16-18 hours) at 2-8° C for serum-free samples. For serum or serum-containing samples, it is recommended to increase incubation time to 16-18 hours at 2-8° C for better sensitivity.

11. Gently remove fluid by vacuum filtration.

12. Wash plate 2 times with 200 µL/well of Wash Buffer, removing Wash Buffer by vacuum filtration between each wash. Remove any excess Wash Buffer from the bottom of the plate using an absorbent pad or paper towels.

13. Add 25 µL of Detection Antibody Cocktail into each well. (Note: allow the Detection Antibody to warm to room temperature, 20-25° C, prior to addition.)

14. Seal, cover with aluminum foil, and incubate with vigorous agitation on a plate shaker at room temperature (20-25° C) for 60 minutes. DO NOT VACUUM AFTER INCUBATION.

15. Add 25 µL Streptavidin-Phycoerythrin to each well containing the 25 µL of Detection Antibody Cocktail.

16. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker for 30 minutes at room temperature (20-25° C).

17. Gently remove all contents by vacuum.

18. Wash plate 3 times with 200 µL/well Wash Buffer, removing Wash Buffer by vacuum filtration between each wash. Wipe any excess buffer on the bottom of plate with a paper towel or tissue.

19. Add 100 µL of Sheath Fluid to all wells. Cover with aluminum foil and resuspend the beads by shaking on a plate shaker for 5 minutes.

20. Run plate on Luminex Instrument. Read 50 beads per bead set in 50 µL sample size.

21. Save the data and evaluate the median fluorescence units using appropriate curve-fitting software. A 5 or 4-parameter logistic method or cubic spline method is recommended.

X. EQUIPMENT SETTINGS

Select the following equipment settings:

Events: 50, per bead
Sample Size: 50 ml
Bead Set: 04 for mMIP-1α
  08 for mGMCSF
  13 for mMCP-1
  15 for mKC
  17 for mRANTES
  19 for mIFNg
  21 for mIL-1b
  26 for mIL-1α
  30 for mG-CSF
  32 for mIP-10
  36 for mIL-2
  52 for mIL-4
  56 for mIL-5
  58 for mIL-6
  59 for mIL-7
  60 for mIL-10
  62 for mIL-12(p70)
  64 for mTNFa
  66 for mIL-9
  68 for mIL-13
  69 for mIL-15
  70 for mIL-17
**Gate (for IS System): 8,000 to 15,000
**Gate (for 1.7 System): 8,000 to 15,000

**These specifications are for the Luminex100 or Luminex200 with software v. 1.7 or IS. Luminex instruments with other software (e.g. Masterplex, Starstation, LiquiChip, Bioplex, LABScan100) would need to follow instrument instructions for gate settings and additional specifications.

XI. QUALITY CONTROLS

The ranges for each analyte in Quality Control 1 and 2 are provided on the card insert or can be located at the Linco Research website www.lincoresearch.com.

XII. ASSAY CHARACTERISTICS

A. Sensitivity (Minimum Detectable Concentrations, minDC)

Cytokine

MinDC (pg/mL)

Cytokine

MinDC (pg/mL)

mMIP-1α

2.8

mIL-4

0.3

mGMCSF

4.6

mIL-5

0.6

mMCP-1

6.3

mIL-6

0.7

mKC

1.2

mIL-10

10.3

mRANTES

0.7

mIL-12p70

3.7

mIFNg

0.7

mTNFα

0.9

mIL-1ß

1.6

mIL-9

10.5

mIL-2

0.8

mIL-13

4.7

mIL-1α

4.4

mIL-17

1.7

mIL-7

3.6

mIP-10

6.0

mIL-15

9.3

mG-CSF

2.3

B. Precision:

Cytokine

Intra-Assay
Precision (CV%)

Inter-Assay
Precision (CV%)

mMIP-1α

3.5

12.4

mGMCSF

1.45

6.4

mMCP-1

5.5

11.7

mKC

12.4

5.7

mRANTES

11.9

3.6

mIFNg

5.3

8.7

mIL-1ß

0.8

12.1

mIL-2

1.9

6.4

mIL-4

9.6

12.3

mIL-5

6.6

7.4

mIL-6

13.8

7.0

mIL-10

9.5

9.7

mIL-12p70

1.6

11.3

mTNFα

7.8

8.9

mIL-9

2.0

12.5

mIL-13

1.7

9.8

mIL-1α

3.7

15.2

mIL-7

8.0

24.0

mIL-15

6.7

21.0

mIL-17

4.3

22.5

mIP-10

10.7

17.3

mG-CSF

7.9

10.6

Intra-assay precision is generated from the mean of the %CV’s from 8 reportable results across two different concentration of cytokines in a single assay. Interassay precision is generated from the mean of the %CV’s from a single reportable result from two different concentrations of cytokine across 4 different assays.

C Accuracy (% Spiked):

Cytokine

Average
%Recovery

Cytokine

Average
%Recovery

mMIP-1α

94.8

mIL-4

110.5

mGMCSF

86.3

mIL-5

82.2

mMCP-1

85.2

mIL-6

81.3

mKC

85.1

mIL-10

94.8

mRANTES

92.7

mIL-12p70

86.2

mIFNg

91.7

mTNFα

96.9

mIL-1ß

89.6

mIL-9

85.8

mIL-2

84.4

mIL-13

85.1

mIL-1α

100.4

mIl-17

81.8

mIL-7

90.0

mIP-10

75.4

mIL-15

97.4

mG-CSF

78.5

Accuracy, defined as the measured cytokine as a percentage of spiked cytokine, was determined by taking the average of 6 levels of cytokine (ranging from 62.5 to 2000 pg/mL) recovered in serum matrix.

D. Dilution Linearity (% Recovery):

Cytokine

Dilution 2X

Dilution 4X

Dilution 8X

mMIP-1α

102.9

98.3

92.2

mGMCSF

96.5

93.5

86.4

mMCP-1

103.9

105.4

99.7

mKC

95.89

117.3

156.6

mRANTES

96.5

107.3

104.8

mIFNg *

---

112.5

131.7

mIL-1ß

99.1

89.2

81.4

mIL-2

109.4

106.0

90.7

mIL-4

150.1

154.6

163.8

mIL-5

99.0

101.4

97.5

mIL-6

95.6

94.1

94.9

mIL-10

81.3

89.9

--

mIL-12p70

98.0

88.2

75.6

mTNFα

108.2

110.3

104.5

mIL-9

96.5

92.4

84.7

mIL-13

88.2

120.9

137.0

mIL-1α

112.64

122.10

160.9

mIL-7

82.3

97.0

104.1

mIL-15

144.65

198.77

-

mIL-17

102.4

105.8

113.8

mIP-10

116.8

126.6

142.9

mG-CSF

105.9

87.2

87.3

* Calculated based on 2X diluted samples.

Dilution Linearity, defined as the cytokine levels measured in diluted samples as a percentage of the neat samples, was determined by measuring cytokine concentrations in 3 independent pools of mouse sera representing 3 strains of mouse, with each pool from 6-8 mice of the same strain. The samples were diluted 1:2, 1:4, and 1:8 in the kit Serum Matrix, and then analyzed in duplicate across 3 assays. The average percent recovery in 3 strains of mice was reported.

XIII. REPLACEMENT REAGENTS

REAGENTS Cat #
Set of two 96-Well Filter Plates with sealers L-PLATE
10x Wash Buffer L-WB
Assay Buffer L-MAB
Serum Matrix LMC-SD
Mouse Cytokine/Chemokine Standard Cocktail LMC-8070
Mouse Cytokine/Chemokine Controls LMC-6070
Mouse Cytokine/Chemokine Detection Antibodies

(Use with Cat # MCB-PMX10)

(Use with Cat # MCB-PMX16)

(Use with Cat. # MCB-PMX22)

LMC-1070-1

LMC-1070-2

LMC-1070-3

Streptavidin-Phycoerythrin

(Use with Cat. # LMC-1070-1)

(Use with Cat. # LMC-1070-2)

(Use with Cat. # LMC-1070-3)

L-SAPE6

L-SAPE4

L-SAPE4

10 Plex Premixed Beads

(mIL-1β, mIL-2, mIL-4, mIL-5, mIL-6, mIL-10, mIL-12, mTNFa, mIFNg, mGM-CSF)

MCB-PMX10
16 Plex Premixed Beads

(mIL-1β, mIL-2, mIL-4, mIL-5, mIL-6, mIL-10, mIL-12, mTNFa, mIFNg, mGMCSF, mMIP-1a, mMCP-1, mKC, mRANTES, mIL-9, mIL-13)

MCB-PMX16
22 Plex Premixed Beads

(mIL-1β, mIL-2, mIL-4, mIL-5, mIL-6, mIL-10, mIL-12, mTNFa, mIFNg, mGMCSF, mMIP-1a, mMCP-1, mKC, mRANTES, mIL-9, mIL-13, mIL-1a, mG-CSF, mIP-10, mIL-7, mIL-15, mIL-17)

MCB-PMX22