RAT ADIPOCYTE LINCOplex KIT 96 Well Plate Assay (CAT # RADPCYT-82K)

Transcription

1 RAT ADIPOCYTE LINCOplex KIT 96 Well Plate Assay (CAT # RADPCYT-82K) I. Intended Use 2 II. Reagents Supplied 2 III. Storage Conditions Upon Receipt 3 IV. Reagent Precautions 3 V. Materials Required But Not Provided 4 VI. Specimen Collection And Storage 4 VII. Technical Guidelines 5 VIII. Preparation of Reagents for Immunoassay 6 IX. Immunoassay Procedure 8 X. Equipment Settings 9 XI. Quality Controls 9 XII. Assay Characteristics 10 XIII. Replacement Reagents 11 XIV. Ordering Information 12 By purchasing this product, which contains fluorescent-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 or or us at info@lincoresearch.com.

2 RAT ADIPOCYTE LINCOplex KIT I. INTENDED USE This multiplex assay kit manufactured by Linco Research is to be used for the simultaneous quantification of the following 7 rat adipokines in any combination: MCP-1, Leptin, IL-1β, IL-6, TNF, PAI-1 (active or total) and Adiponectin. This kit can be used for the analysis of the above analytes in tissue extract or cell/tissue culture samples. Note that Active PAI-1 and Total PAI-1 cannot be run together in the same assay. This kit is for research purposes only. II. REAGENTS SUPPLIED A. Antibody-Immobilized Beads Dilution is required before the assay. The following beads are available for your customized orders: #13-MCP-1 #16-Leptin #34-IL-1β #38-IL-6 #51-Adiponectin #77-TNF #86-PAI-1 (active) #89-PAI-1 (total) Quantity: 200 l antibody-immobilized beads per bottle Note that Active PAI-1 and Total PAI-1 cannot be run together in the same assay. B. Rat Adipokine Standard Cocktail 1 vial containing rat adipokine standard cocktail, lyophilized Quantity: 1 vial C. Rat Adipokine Controls Control 1 1 vial containing adipokine cocktail, lyophilized Control 2 1 vial containing adipokine cocktail, lyophilized Quantity: 1 vial each control D. Rat Adipocyte Detection Antibodies 1 bottle containing a cocktail of biotinylated detection antibodies in Assay Buffer Quantity: 5.5 ml/bottle RADPCYT-82K Rev. 08/01/06 Linco Research 2

3 II. REAGENTS SUPPLIED (continued) 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. Freeze reconstituted standards and controls at -20 C. Avoid multiple (>2) freeze/thaw cycles. DO NOT FREEZE Antibody-Immobilized Beads, Bead Diluent, 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. RADPCYT-82K Rev. 08/01/06 Linco Research 3

4 V. MATERIALS REQUIRED BUT NOT PROVIDED A. Reagents Luminex Sheath Fluid (Luminex Catalogue # ) B. Instrumentation/Materials 1. Adjustable Pipettes with Tips capable of delivering 10 µl to 1000 µl 2. Multichannel Pipettes capable of delivering 10 µl to 200 µl 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. Luminex Instrument VI. SPECIMEN COLLECTION AND STORAGE A total of 25 µl per well tissue/cell extract or supernatant samples collected from adipose/adipocytes are used. Appropriate sample dilution with adipokine-free culture media may be required. RADPCYT-82K Rev. 08/01/06 Linco Research 4

5 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. 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 µl of buffer in 5 seconds (equivalent to < 100 mmhg). E. After hydration, all standards and controls must be transferred to polypropylene tubes. F. The standards prepared by serial dilution must be used within 1 hour of preparation. Discard any unused standards except the highest stock standard which may be stored at -20 C for 1 month and at -80 C for greater than one month. G. Any unused mixed Antibody-Immobilized Beads should be discarded after use. 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. Prior to reading, agitate the plate on the plate shaker for 1-10 minutes. Long delay in reading the plate may result in decreased signals and sensitivities for some analytes. J. 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 rpm in a 0.3 cm orbit. 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 to 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 dilution is required, samples should be diluted in adipokine-free culture media. RADPCYT-82K Rev. 08/01/06 Linco Research 5

6 VIII. PREPARATION OF REAGENTS FOR IMMUNOASSAY A. Preparation of Rat Adipokine Standard Cocktail 1). Prior to use, reconstitute the Rat Adipokine Standard Cocktail with 250 l Deionized Water to give the highest stock concentration of standard. Invert the vial several times to mix. Allow the vial to set for 5 minutes to make sure that the standards are completely reconstituted, and then transfer the standard to a polypropylene microfuge tube labeled as Standard 7. The unused portions of this standard may be stored at -20 for up to one month. 2). Preparation of Working Standards Label six polypropylene microfuge tubes Standard 1, Standard 2, Standard 3, Standard 4, Standard 5, and Standard 6. Add 150 µl of Assay Buffer to each of the six tubes. Prepare 4 time serial dilutions by adding 50 µl of the Standard 7 to the Standard 6 tube, mix well and transfer 50 µl of the Standard 6 tube to the Standard 5 tube, mix well and transfer 50 µl of the Standard 5 tube to the Standard 4 tube, mix well and transfer 50 µl of the Standard 4 tube to the Standard 3 tube, mix well and transfer 50 µl of the Standard 3 tube to the Standard 2 tube, mix well and transfer 50 µl of the Standard 2 tube to the Standard 1 tube and mix well. The 0 standard (Background) will be the Assay Buffer. Standard Dilution Original Standard 7 Volume of Deionized Water to Add Volume of Standard to Add 250 µl water 0 Standard Dilution Volume of Assay Buffer to Add Volume of Standard to Add Standard µl 50 µl of Standard 7 Standard µl 50 µl of Standard 6 Standard µl 50 µl of Standard 5 Standard µl 50 µl of Standard 4 Standard µl 50 µl of Standard 3 Standard 1 (optional) 150 µl 50 µl of Standard 2 RADPCYT-82K Rev. 08/01/06 Linco Research 6

7 VIII. PREPARATION OF REAGENTS FOR IMMUNOASSAY (continued) The serial dilutions result in the following concentrations of standards. Standard Dilution IL-1, TNF-, MCP-1 Standard (pg/ml) Leptin, IL-6, PAI-1 Standard (pg/ml) Adiponectin (pg/ml) Standard 7 20,000 50, ,000 Standard 6 5,000 12, ,000 Standard 5 1,250 3,125 31,250 Standard ,813 Standard ,953 Standard Standard B. Preparation of Controls Before use, reconstitute Rat Adipokine Control 1 and Rat Adipokine Control 2 with 250 l Deionized water. Invert the vials several times to mix and allow the vials to set for about 5 minutes. Transfer each Control to an appropriately labeled polypropylene microfuge tube. Unused portions may be stored at -20 C for up to one month. C. 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. D. Preparation of Antibody-Immobilized Beads Make the bead mixture right before setting up the assay. Early preparation of the bead mixture (>1 day) should be avoided. Sonicate each antibody-bead bottle for 30 seconds, vortex for 1 minute. Add 0.15 ml from each antibody bead bottle to the Mixing Bottle and bring final volume to 3 ml with Assay Buffer. Unused portions of the bead mixture should be discarded after use. Left-over individual beads can be stored at 2-8 C for up to one month. Example 1: when using six antibody-immobilized beads, add 0.15 ml from each of the six bead sets to the mixing bottle and add 2.1 ml assay buffer. Example 2: when using four antibody-immobilized beads, add 0.15 ml from each of the four bead sets to the mixing bottle and add 2.4 ml Assay Buffer. RADPCYT-82K Rev. 08/01/06 Linco Research 7

8 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) 1, 2, 3, 4, 5, 6, 7, Control 1 and 2, 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. Pre-wet 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 PLATES). 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 Adipokine free culture media to the Background, Standards, and Control wells. 8. Add 25 µl of Sample into the appropriate wells. 9. Vortex Bead Mix Bottle 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 overnight (16-18 hours) at 2-8 o C. After overnight incubation, it is important to allow the plate and reagents to warm to room temperature (20-25 C) before continuing with the assay. 11. Gently remove fluid by vacuum. 12. Wash plate 3 times with 200 µl/well of Wash Buffer, removing Wash Buffer by vacuum filtration between each wash. Remove any excess Assay Buffer from the bottom of the plate by blotting on an absorbent pad or paper towels. 13. Pipet 50 µl of Detection Antibody Cocktail into each well. (Note: It is important to allow the Detection Antibody to warm to room temperature, C, prior to addition.) 14. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker for 1 hour at room temperature. DO NOT VACUUM AFTER INCUBATION. 15. Add 50 µl Streptavidin-Phycoerythrin to each well containing the 50 µl of Detection Antibody Cocktail. (Note: allow the SAPE to warm to room temperature prior to addition.) 16. Seal, cover with aluminum foil, and incubate with agitation on a plate shaker for 30 minutes at room temperature. RADPCYT-82K Rev. 08/01/06 Linco Research 8

9 IX. IMMUNOASSAY PROCEDURE (continued) 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. Remove any excess Assay Buffer from the bottom of the plate by blotting on an absorbent pad or paper towels. 19. Add 100 µl of Sheath Fluid to all wells. Seal, cover with aluminum foil and resuspend the beads on a plate shaker for 5 minutes. 20. Read plate on Luminex Instrument. Read 50 beads per bead set in 50 µl sample size. 21. Save and evaluate the median data using a 5-parameter or spline fit data reduction. X. EQUIPMENT SETTINGS Select the following equipment settings: Events: 50 per bead Sample Size: 50 µl Bead Set: 13 for MCP-1 16 for Leptin 34 for IL-1β 38 for IL-6 51 for Adiponectin 77 for TNF 86 for PAI-1 (active) 89 for PAI-1 (total) **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 v1.7 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 RADPCYT-82K Rev. 08/01/06 Linco Research 9

10 XII. ASSAY CHARACTERISTICS A. Sensitivity (MinDC, pg/ml) MCP Leptin 2.5 IL IL Adiponectin 32.7 TNF- 1.1 PAI-1 (active) 14.7 PAI-1 (total) 12.5 B. Accuracy (recovery in buffer) MCP-1 99% Leptin 97% IL-1 96% IL-6 89% Adiponectin 99% TNF- 99% PAI-1 (active) 92% PAI-1 (total) 117% C. Precision Intra-assay variation (%CV) <8% Inter-assay variation (%CV) <20% D. Cross-Reactivity The antibody pairs used in this assay are specific and there is no significant cross-reaction within this panel. RADPCYT-82K Rev. 08/01/06 Linco Research 10

11 XIII. REPLACEMENT REAGENTS Rat Adipokine Standard Cocktail Rat Adipokine Quality Controls 1 and 2 Rat Adipocyte Detection Antibodies Streptavidin-Phycoerythrin Assay Buffer Set of two 96-Well Filter Plates with sealers 10X Wash Buffer Concentrate MCP-1 Beads Leptin Beads IL-1β Beads IL-6 Beads Adiponectin Beads TNF- Beads PAI-1 (Active) Beads PAI-1 (Total) Beads LRA-8081 LRA-6081 LRAC-1082 L-SAPE LE-ABGLP L-PLATE L-WB RMCP-1 RME-LPTN RIL-1B RIL-6 HA-ADPN RTNF-A HA-PAI1 RA-PAI1 RADPCYT-82K Rev. 08/01/06 Linco Research 11

12 XIV. ORDERING INFORMATION A. To place an order: To assure the clarity of your custom Adipokine kit order, please FAX the following information to our customer service department: 1. Your name, telephone and/or fax number 2. Customer account number 3. Shipping and billing address 4. Purchase order number 5. Catalog number and description of product 6. Quantity of kits 7. Selection of LINCOplex Adipokine analytes FAX: (636) Toll Free US: (866) (636) MAIL ORDERS: Linco Research 6 Research Park Drive St. Charles, Missouri U.S.A. For International Customers: To best serve our international customers, it is LINCO s policy to sell our products through a network of distributors. To place an order or to obtain additional information about LINCO products, please contact your local distributor. B. Conditions of Sale All products are for research use only. They are not intended for use in clinical diagnosis or for administration to humans or animals. All products are intended for in vitro use only. C. Material Safety Data Sheets (MSDS) Material Safety Data Sheets for Linco Research products may be ordered by fax or phone. RADPCYT-82K Rev. 08/01/06 Linco Research 12

13 WELL MAP A 0 Standard (Background) Standard 4 QC-1 Control B 0 Standard (Background) Standard 4 QC-1 Control C Standard 1 Standard 5 QC-2 Control D Standard 1 Standard 5 QC-2 Control E Standard 2 Standard 6 F Standard 2 Standard 6 G Standard 3 Standard 7 H Standard 3 Standard 7 RADPCYT-82K Rev. 08/01/06 Linco Research 13

14 Assay Method for Rat Adipocyte LINCOplex (RADPCYT-82K) Kit Well # Well Identification Assay Buffer Assay Buffer Standard/ Control/Sample Appropriate Media Mixed Beads Detection Antibody SA-PE Sheath Fluid 1A, 1B 0 Standard (Background) 200 µl 25 µl - 25 µl 25 µl 50 µl 50 µl 100 µl Seal, Agitate, Incubate 10 minutes at Room Temperature. Remove Assay Buffer by Vacuum 1C, 1D Standard 1-25 µl 25 µl 1E, 1F Standard 2-25 µl 25 µl 1G, 1H Standard 3-25 µl 25 µl 2A, 2B Standard 4-25 µl 25 µl 2C, 2D Standard 5-25 µl 25 µl 2E, 2F Standard 6-25 µl 25 µl 2G, 2H Standard 7-25 µl 25 µl 3A, 3B Control 1-25 µl 25 µl 3C, 3D Control 2-25 µl 25 µl 3E, 3F Sample 25 l 25 µl - 3G, 3H Sample 25 µl 25 µl - Seal, Agitate, Incubate overnight (16-18 hrs) at 2-8 o C Wash 3X with 200 µl Wash Buffer Seal, Agitate, Incubate 1 hour at Room Temperature. Seal, Agitate, Incubate 30 minutes at Room Temperature. Wash 3X with 200 µl Wash Buffer 4A, 4B Sample 25 µl 25 µl - 4C, 4D Final Sample Sample Sample 25 µl 25 µl - 25 µl 25 µl - RADPCYT-82K Rev. 08/01/06 Linco Research 14