MOUSE ADIPOCYTE 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 Luminex100.
This multiplex assay kit manufactured by Linco Research is to be used for the simultaneous quantification of the following 7 mouse adipokines in any combination: Leptin, Resistin, Adiponectin, IL-6, TNFα, MCP-1, and PAI-1 (total).
This kit is for research purposes only.
A. Antibody-Immobilized Beads
The following beads are available for your customized orders:
#13-Mouse MCP-1
#16-Leptin
#58-Mouse IL-6
#64-Mouse TNFα
#75-Mouse PAI-1
#78-Mouse Resistin
#81-Mouse Adiponectin
Mix beads and dilute with Assay Buffer HENDO-65K as described in Section VIII. D.
Quantity: 200μl antibody-immobilized beads per bottle
B. Mouse Adipokine Standard Cocktail
1 vial containing mouse adipokine standard cocktail, lyophilized
Quantity: 1 vial
C. Mouse Adipokine Controls
Control I – 1 vial containing adipokine cocktail, lyophilized
Control II – 1 vial containing adipokine cocktail, lyophilized
Quantity: 1 vial each Control
D. Mouse Adipocyte Detection Antibodies
1 bottle containing a cocktail of biotinylated detection antibodies in Assay Buffer
Quantity: 5.5 mL/bottle
E. Streptavidin-Phycoerythrin
1 bottle containing Streptavidin-Phycoerythrin in Assay Buffer
Quantity: 5.5 mL/bottle
F. Assay Buffer
PBS with 0.08% Sodium Azide, 0.05% Tween 20, Protease Inhibitor and 1% BSA, pH 6.8
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. Mixing Bottle
Quantity: 1 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, 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. 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 10 m l to 1000 m l
2. Multichannel Pipettes capable of delivering 10 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 #MAVMO960R, or equivalent)11. Luminex100 by Luminex Corporation
VI. SPECIMEN COLLECTION AND STORAGE
A 10 m l per well of tissue/cell extract or supernatant samples are used. Appropriate sample dilution with Assay Buffer may be required.
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.
A. The Antibody-Immobilized Beads are light sensitive, so the assay plate containing beads must be covered with aluminum foil during all incubation steps.
B. The bottom of the Microtiter Filter Plate should not be in contact with any absorbent material during assay set-up or incubation times. Set the plate on a plate cover or any other plate holder to raise the plate from the surface.
C. After the wash steps, dry the bottom of the Microtiter Filter Plate by using paper towels or absorbent pads to prevent any leakage due to capillary action.
D. 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)
E. The standards prepared by serial dilution must be used within 1 hour of preparation. Discard any unused standards except the 50,000 pg/mL stock standard which may be stored at £ -20° C for 1 month and at £ -80° C for greater than one month.
F. Any unused mixed Antibody-Immobilized Beads may be stored in the bead mix bottle at 2-8° C for up to one month.
G. 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.
H. If the plate cannot be read on the same day as the assay was finished, do the final bead suspension in 100 ml Sheath Fluid, 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.
I. 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 in a 0.3 cm orbit.
J. 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.
K. 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 Mouse Adipokine Standard Cocktail
Reconsititute the Mouse Adipokine Standard Cocktail with 250 μl deionized water to give a 50,000 pg/mL concentration of standard stock. Allow the vial to set for about 5 minutes, invert the vial several times to mix. Vortex and mix well. Transfer the standards to a microfuge tube labeled 50,000. Label six polypropylene microfuge tubes 12,500, 3,125, 781.3, 195.3, 48.8, and 12.2. Add 150 ml of Assay Buffer to each of the six tubes. Perform 4 times serial dilutions by adding 50 ml of the 50,000 (pg/mL) standard to the "12,500" tube, mix well and transfer 50 ml of the "12,500" standard to the "3,125" tube, mix well and transfer 50 ml of the "3,125" standard to the "781.3" tube, mix well and transfer 50 μl of the “781.3" standard to "195.3" tube, mix well and transfer 50 ml of the "195.3" pg/mlstandard to the "48.8", mix well and transfer 50 ml of the "48.8" pg/mlstandard to the "12.2" tube. The 0 standard (Background) will be Assay Buffer.