FMAT Blue Dye-Labeled Chemokines Applied Biosystems 8200 Cellular Detection System

Transcription

1 FMAT Blue Dye-Labeled Chemokines Applied Biosystems 8200 Cellular Detection System Protocol

2 Copyright 2006, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. TRADEMARKS: Applera, Applied Biosystems, AB (Design), FMAT, and FMAT Blue are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. All other trademarks are the sole property of their respective owners. Part Number Rev. A 09/2006

3 Contents Front Matter Preface v Safety v How to Obtain More Information x How to Obtain Support x Overview Storage Conditions Required Materials Prepare the FMAT Blue Dye-Labeled Chemokine Prepare the Unlabeled Chemokine Perform the Assay iii

4 Contents iv

5 Preface This preface covers: Safety v How to Obtain More Information x How to Obtain Support x Safety Safety Alert Words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards. Each alert word IMPORTANT, CAUTION, WARNING, DANGER implies a particular level of observation or action, as defined below. Definitions IMPORTANT! Indicates information that is necessary for proper instrument operation, accurate chemistry kit use, or safe use of a chemical. Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury. Indicates an imminently hazardous situation that, if not avoided, will result in death or serious injury. This signal word is to be limited to the most extreme situations. v

6 Preface vi Chemical Hazard Warning Chemical Safety Guidelines About MSDSs Obtaining MSDSs CHEMICAL HAZARD. Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury, illness, or death. To minimize the hazards of chemicals: Read and understand the Material Safety Data Sheets (MSDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. (See About MSDSs on page vi.) Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the MSDS. Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturer s cleanup procedures as recommended in the MSDS. Comply with all local, state/provincial, or national laws and regulations related to chemical storage, handling, and disposal. Chemical manufacturers supply current Material Safety Data Sheets (MSDSs) with shipments of hazardous chemicals to new customers. They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated. MSDSs provide the safety information you need to store, handle, transport, and dispose of the chemicals safely. Each time you receive a new MSDS packaged with a hazardous chemical, be sure to replace the appropriate MSDS in your files. The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day. To obtain MSDSs: 1. Go to 2. In the Search field of the MSDS Search page: a. Type in the chemical name, part number, or other information that you expect to appear in the MSDS of interest.

7 Safety b. Select the language of your choice. c. Click Search. 3. To view, download, or print the document of interest: a. Right-click the document title. b. Select: Open To view the document Save Target As To download a PDF version of the document to a destination that you choose Print Target To print the document 4. To have a copy of an MSDS sent by fax or , in the Search Results page: a. Select Fax or below the document title. b. Click RETRIEVE DOCUMENTS at the end of the document list. c. Enter the required information. d. Click View/Deliver Selected Documents Now. Note: For the MSDSs of chemicals not distributed by Applied Biosystems, contact the chemical manufacturer. Chemical Waste Hazards HAZARDOUS WASTE. Refer to Material Safety Data Sheets and local regulations for handling and disposal. CHEMICAL WASTE HAZARD. Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury, illness, or death. CHEMICAL STORAGE HAZARD. Never collect or store waste in a glass container because of the risk of breaking or shattering. Reagent and waste bottles can crack and leak. Each waste bottle should be secured in a low-density polyethylene safety container with the cover fastened and the handles locked in the upright position. Wear appropriate eyewear, clothing, and gloves when handling reagent and waste bottles. vii

8 Preface Chemical Waste Safety Guidelines Waste Disposal To minimize the hazards of chemical waste: Read and understand the Material Safety Data Sheets (MSDSs) provided by the manufacturers of the chemicals in the waste container before you store, handle, or dispose of chemical waste. Provide primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.) Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing). For additional safety guidelines, consult the MSDS. Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood). For additional safety guidelines, consult the MSDS. Handle chemical wastes in a fume hood. After emptying the waste container, seal it with the cap provided. Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local, state/provincial, or national environmental and health regulations. If potentially hazardous waste is generated when you operate the instrument, you must: Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory. Ensure the health and safety of all personnel in your laboratory. Ensure that the instrument waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations. IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply. viii

9 Safety Biological Hazard Safety BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective equipment, which includes but is not limited to: protective eyewear, face shield, clothing/lab coat, and gloves. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Individuals should be trained according to applicable regulatory and company/institution requirements before working with potentially infectious materials. Read and follow the applicable guidelines and/or regulatory requirements in the following: U.S. Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories (stock no ; Occupational Safety and Health Standards, Bloodborne Pathogens (29 CFR ; nara/cfr/waisidx_01/ 29cfr1910a_01.html). Your company s/institution s Biosafety Program protocols for working with/handling potentially infectious materials. Additional information about biohazard guidelines is available at: ix

10 Preface How to Obtain More Information Related Documentation Send Us Your Comments For more information, refer to the Applied Biosystems 8200 Cellular Detection System User s Guide (PN ). Applied Biosystems welcomes your comments and suggestions for improving its user documents. You can your comments to: techpubs@appliedbiosystems.com IMPORTANT! The address above is only for submitting comments and suggestions relating to documentation. To order documents, download PDF files, or for help with a technical question, go to then click the link for Support. (See How to Obtain Support below). How to Obtain Support For the latest services and support information for all locations, go to then click the link for Support. At the Support page, you can: Search through frequently asked questions (FAQs) Submit a question directly to Technical Support Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition, the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities. x

11 Overview This protocol provides suggested procedures for performing a chemokine competition assay with the Applied Biosystems 8200 Cellular Detection System. Applied Biosystems recommends these procedures as a starting point for specific chemokine competition assays. For your specific assays, you may need to optimize parameters such as labeled and unlabeled chemokine concentrations, cell density, and so on. About the 8200 System About FMAT Blue Dye-Labeled Chemokines The Applied Biosystems 8200 Cellular Detection System uses the fluorescence-based FMAT system technology to screen drug candidates that modulate the interaction of chemokines and their receptors. Note: This protocol assumes users can operate the 8200 System with a minimum level of proficiency. For more information on operating the 8200 System, refer to the Applied Biosystems 8200 Cellular Detection System User s Guide. Applied Biosystems provides multiple labeled chemokines with the FMAT Blue dye for use on the 8200 System or other fluorescencebased platforms. At least 18 chemokine receptors have been characterized (designated by chemokine subfamily name): Ten CC-receptors (CCR1 through CCR10) Six CXC-receptors (CXCR1 through CXCR6) One C-receptor (XCR1) One CX 3 C-receptor (CX 3 CR1) 1

12 Overview In the figure below, these 18 receptors are shown inside the circle; known ligands are shown outside the circle. Note: CXCL4, CXCL14, CXCL15, CCL6, and CCL18 are not shown because their receptors are unknown. Note: Figure adapted from J. Immunol. Methods, 2002; 262:1-3. Excitation/Emission of FMAT Blue Dye-Labeled Chemokines The FMAT Blue dye-labeled chemokines have an: Excitation wavelength of 633 nm Emission wavelength peak at 650 nm (maximum) Workflow This protocol describes how to: Prepare the FMAT Blue dye-labeled chemokine (page 5). Prepare the unlabeled chemokine (page 7). Perform the assay (page 8). 2

13 Storage Conditions Technical Support For technical problems related to the 8200 System and the FMAT Blue dye-labeled chemokines, contact Applied Biosystems Technical Support: Phone 1. Dial Enter the product part number, followed by the asterisk key (*). 3. Press 6. Fax Web site For international customers and for additional technical support contact information, see How to Obtain Support on page x of the Preface. Storage Conditions Dried Chemokine Reconstituted Chemokine Store the dried FMAT Blue dye-labeled chemokine at 20 C until ready for use. Store the reconstituted FMAT Blue dye-labeled chemokine at 20 C in polypropylene Eppendorf tubes at the recommended solution and concentration (see page 5 for reconstitution details) until ready for use. Note: Avoid multiple freeze-thaw cycles. 3

14 Required Materials Required Materials Item FMAT Blue dye-labeled chemokine Benchtop centrifuge Acetonitrile (ACN) Source Applied Biosystems (See Applied Biosystems Web site for part numbers.) Major Laboratory Supplier (MLS) MLS 0.1% trifluoroacetic acid (TFA) in deionized water (dih 2 O) MLS Vortex mixer Polypropylene Eppendorf tubes Unlabeled dried chemokine of interest Phosphate-buffered saline (PBS)/0.1%bovine serum albumin (BSA) Hepes Binding Buffer: 10-mM Hepes, ph 7.4; 2.5-mM CaCl 2 ; 140- mm NaCl, 0.5% (w/v) BSA; 3-mM MgCl 2 ; 0.01% Na Azide Cells expressing the appropriate chemokine receptor 96-well FMAT plate 384-well FMAT plate MLS MLS User-supplied MLS MLS User-supplied Applied Biosystems (PN ) Applied Biosystems (PN ) Hepes Binding Buffer is the recommended buffer for performing chemokine receptor binding assays. Occasionally, the ph and ion concentrations may need to be altered to optimize the assay. Some chemokines naturally exhibit nonspecific binding to polystyrene plates. To reduce nonspecific binding in such cases, Applied Biosystems recommends performing the binding assay on a low-bind plate, such as the Corning 96 Well Black with Clear Flat Bottom Polystyrene NBS Microplate (PN 3651 or 3991) from Corning Life Sciences. Note: For the Material Safety Data Sheet (MSDS) of any chemical not distributed by Applied Biosystems, contact the chemical manufacturer. Before handling any chemicals, refer to the MSDS provided by the manufacturer, and observe all relevant precautions. 4

15 Prepare the FMAT Blue Dye-Labeled Chemokine Prepare the FMAT Blue Dye-Labeled Chemokine Pellet the Dried Chemokine Reconstitute the Chemokine Before you reconstitute the dried FMAT Blue dye-labeled chemokine: 1. Spin the dried chemokine at 1300 rpm in a benchtop centrifuge for 60 seconds. 2. Inspect to make sure the chemokine has been pelleted. If no pellet is observed, re-spin as in step 1 until a visible pellet is formed. 1. Prepare a 20% ACN/0.08% TFA reconstitution solvent by combining: 2mL of ACN 8 ml of 0.1% TFA in dih 2 O 2. Prepare a 50-µM stock solution: a. Add 2 µl of the reconstitution solvent per 100 pmol of the pelleted chemokine. For example: Pelleted Chemokine Reconstitution Solvent (µl) Chemokine Final Conc. (µm) 100-pmol aliquot nmol aliquot b. Briefly vortex. c. Spin at 1300 rpm in a benchtop centrifuge for 60 seconds. d. Inspect the 50-µM stock solution to make sure the chemokine is dissolved into solution. 3. At this point, you can: Dilute and use the reconstituted 50-µM chemokine stock solution immediately (see page 6). Or Store the reconstituted 50-µM chemokine stock solution at 20 C until ready for use. Note: Avoid multiple freeze-thaw cycles. 5

16 Prepare the FMAT Blue Dye-Labeled Chemokine Dilute the Reconstituted Chemokine 1. Dilute the reconstituted 50-µM chemokine stock solution to 3-fold the chemokine s binding affinity and Kd concentration (3 Kd) in a polypropylene Eppendorf tube. Note: You can calculate the Kd value by performing a Saturation Binding Analysis experiment on the 8200 System. You can also find the Kd value from literature. Note: You need approximately 3 ml of the 3 Kd diluted FMAT Blue dye-labeled chemokine for each 96-well plate. 2. Use the 3 Kd solution immediately (see Perform the Assay on page 8). 6

17 Prepare the Unlabeled Chemokine Prepare the Unlabeled Chemokine 1. Prepare a 10-µM stock solution of the unlabeled dried chemokine of interest: Add 12.5 µl of PBS/0.1% BSA per microgram of chemokine. 2. Prepare ten ~3-fold serial dilutions of the unlabeled chemokine by adding the 10-µM stock solution to the Hepes Binding Buffer, as indicated below. Working Conc. (nm) Stock Dilution Buffer (µl) Final In-Well Conc. (nm) (Using 30 µl/well) µl of 10-µM stock µl of 300-nM stock µl of 90-nM stock µl of 30-nM stock µl of 3-nM stock µl of 3-nM stock µl of 0.9-nM stock µl of 0.3-nM stock µl of 0.09-nM stock µl of 0.03-nM stock Use the diluted unlabeled chemokine immediately (see Perform the Assay on page 8). Dispose of any unused chemokine appropriately. 7

18 Perform the Assay Perform the Assay Perform the assay using the IC50 Assay Curve protocol for: Adherent cells (below) Or Suspension cells (page 9) Protocol for Adherent Cells 1. Remove adherent cells from the cell culture flasks, then dilute to cells/ml in cell culture medium. 2. Add 100 µl of the cell suspension to each well of a 96-well plate. 3. Incubate the plate overnight at 37 C, 5% CO 2. During incubation the cell number approximately doubles to Remove the cell culture medium, then wash twice with 100 µl of Hepes Binding Buffer. 5. Add 30 µl of Hepes Binding Buffer to each well. 6. Add 30 µl of each dilution of the unlabeled chemokine (from page 7) to the wells indicated below. 100 nm 30 nm 10 nm 3 nm 1 nm 0.3 nm 0.1 nm 0.03 nm 0.01 nm nm Buffer blank Buffer blank 7. Add 30 µl of the 3 Kd solution (from page 6) to each well. 8. Incubate for 1 to 2 hours at 37 C. 9. Scan the plate and analyze the data (see page 9). 8

19 Perform the Assay Protocol for Suspension Cells 1. In Hepes Binding Buffer, prepare a suspension of cells expressing the receptor of interest at cells/ml. 2. To a 96-well plate, add 30 µl of each dilution of the unlabeled chemokine (from page 7) to the wells indicated below. 100 nm 30 nm 10 nm 3 nm 1 nm 0.3 nm 0.1 nm 0.03 nm 0.01 nm nm Buffer blank Buffer blank 3. Add 30 µl of the 3 Kd solution (from page 6) to each well. 4. Add 30 µl of the suspension cells to each well for a final concentration of ~10,000 cells/well. 5. Incubate for 1 to 2 hours at 37 C. 6. Scan the plate and analyze the data (see below). Scan the Plate and Analyze the Data Antibody Assay Using the 8200 System: 1. Scan the plate. 2. Analyze the data using the one-color, one-population IC50 assay set up. See the Applied Biosystems 8200 Cellular Detection System User s Guide for details on scanning the plate and analyzing the data. You can perform a similar assay using either adherent or suspension cells by substituting an antibody directed against the receptor in place of the unlabeled chemokine. 9

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