TECHNICAL DATA SHEET AequoZen® Caution: For Laboratory Use. A research reagent for research purposes only Human Chemokine CXCR2 Receptor, -Irradiated Frozen Cells Product No.: ES-145-AF Lot No.: 1003 Material Provided Cells: 1 x 1 mL frozen aliquot Format: ~10 x 10 cells/mL in Ham’s F12, 10% FBS with 10 % DMSO 6 Product Information Cellular Background: CHO-K1 Parental Frozen Cells(control) : A2 (Cat # ES-000-A2F) Frozen Cells Info: Frozen recombinant, irradiated CHO-K1 cells expressing mitochondrially-targeted Aequorin, Gα16 and the human Chemokine CXCR2 receptor. DNA Sequence: Identical to coding sequence of GenBank M73969.1. Corresponding Protein Sequence: Identical to GenBank NP_001548.1. Storage Conditions: Store in liquid nitrogen (vapor phase) immediately upon receipt, or ® maximum 15 days at -80°C. AequoZen is designed for single use only. Do not refreeze. Quality Control ® EC50 for a reference agonist is determined using an AequoScreen assay (Figure 1). Mycoplasma test is performed ® using MycoAlert Mycoplasma detection kit. We certify that these results meet our quality release criteria. IL8 (EC50): 1.63 nM Mycoplasma: This cell line tested negative for Mycoplasma. TDS-ES-145-AF-05 Page 1 of 5 Recommended Thawing Conditions and Handling of Frozen Cells Carefully follow instructions below to obtain the expected results. Most Frozen cells are intended to be assayed immediately upon thawing. Exceptionally, where specified, some frozen cell products require an overnight incubation in Cell Medium to enable them to perform optimally. The recommended media catalogue number and supplier reference information are listed in this Product Technical Data Sheet (last page). Media composition is specifically defined for each cell type and receptor. The use of incorrect media or component substitutions can lead to altered product performance. Additionally, the instructions for the preparation of ligands must be carefully followed to avoid ligand precipitation, degradation or adsorption. Inappropriate preparation may result in a non-representative pharmacology. The complete thawing procedure must not exceed 30 min. Cell viability below 90% upon thawing may indicate that the Frozen cells were affected by incorrect thawing procedure and may yield to lower performance. Ensure the cells are not clumped and are evenly distributed in the assay plates. Gently pipet up and down if cells are clumped before dispensing the cells. Frozen cells cannot be re-frozen. ® Assay Medium (for immediate thaw and use): AequoScreen Assay Buffer (see below) Cell Medium (for overnight incubation prior to use): Ham's F-12, 10% FBS Thawing Cells: Using appropriate personal protective equipment, rapidly place the frozen aliquot in a 37°C water bath (do not submerge) until its content is thawed completely. Immediately remove from water bath, spray aliquot with 70% ethanol and wipe excess. Under aseptic conditions using a sterile pipette, gently resuspend the cells in the cryovials and transfer content to a sterile centrifuge tube containing 10 mL of the Assay or Cell Medium prewarmed to 37°C, and centrifuge (150 x g, 5 min.). Do not exceed the recommended centrifugal force. Discard supernatant using a sterile pipette. Gently resuspend cell pellet in 5 mL of appropriate pre-warmed medium by gently pipetting up and down to break up any clumps. For immediate use, dilute cells to recommended cell density in Assay Medium. For an overnight incubation step, plate the cells in Cell Medium in a T75 cm culture flask. Incubate overnight at 37°C in a humidified atmosphere with 5% CO2. Most cells will adhere but they will not grow, due to the gamma-irradiation process. To harvest cells, under aseptic conditions, remove media, rinse with 4.5 mL of calcium and magnesium-free PBS, add 4.5 mL Versene or calcium and magnesium-free PBS/0.5 mM EDTA, and incubate at room temperature until cells detach (do not exceed 5-10 minutes). Add 9 mL of Assay Medium, collect the cells, centrifuge (150 x g, 5 min) and resuspend in Assay Medium to the recommended cell density. 2 Recommended Cell Density per Assay Point (MicroLumat): 25 000 cells/well Optimal cell density per assay point will depend on the sensitivity of the reader used. The values given here are the ones determined for using the cells on a MicroLumat Reader, but may need optimization when using another reader. Reducing the coelenterazine concentration and loading time in the AequoScreen assay will still result in some signal, but is expected to result in decreased signal intensity and/or stability, so we do not recommend reducing these parameters. Temperature should remain below 25°C during the coelenterazine loading of the cells, and until using the cells for the readings. Excessive heating by the cell stirrer for example will result in signal loss. ® TDS-ES-145-AF-05 Page 2 of 5 Emitted Light (RLU) Typical Product Data 600000 EC50 Agonist IL8 GRO alpha GRO béta 800000 IL8 400000 % of Digitonin (M) 8.1 x 10 response -10 64 56 52 GROα 2.6 x 10 -9 GROβ 4.5 x 10 -9 200000 0 -14 -13 -12 -11 -10 -9 -8 -7 -6 Log [Ligand], M ® Figure 1: Agonist Response in AequoScreen assay An agonist dose-response experiment was performed in 96-well format using 25 000 cells/well. Luminescence was measured with the MicroLumat. Data from a representative experiment are shown. The Z’-factor was calculated for IL8 with at least 16 background and 16 maximal signal points (Z’= 0.90). Emitted Light (RLU) 600000 400000 Antagonist SB225002 SB 225002 200000 0 -15.0 -12.5 -10.0 -7.5 -5.0 IC50 (M) 5.1 x 10 -7 -2.5 Log [SB225002], M ® Figure 2: Antagonist Response in AequoScreen assay An antagonist dose-response experiment was performed in 96-well format using 25 000 cells/well. The reference agonist used was IL8, at a final concentration equivalent to the EC80. Luminescence was measured with the MicroLumat. Data from a representative experiment are shown. TDS-ES-145-AF-05 Page 3 of 5 AequoScreen® Assay Procedure (MicroBeta® JET) Assay Buffer: DMEM / HAM’s F12 with HEPES, without phenol red (Invitrogen # 11039-021) + 0.1 % protease-free BSA (from 10% solution sterilized by filtration at 0.22 µm). Store at 4°C. Coelenterazine h: To prepare a 500 µM Coelenterazine h stock solution, solubilize 250 µg of Coelenterazine h (Promega # S2011 or Invitrogen # C6780) in 1227 µL methanol. Store at -20°C in the dark. Digitonin: To prepare a 50 mM Digitonin stock solution, dissolve 1 g of Digitonin (Sigma # 37006) in 16.27 mL of DMSO. Aliquot and store at -20°C. 1. Cell preparation: Resuspend thawed cells prepared as exposed above in Assay Buffer at a 5 concentration of 3x10 cells/mL. 2. Coelenterazine Loading: Under sterile conditions, add “Coelenterazine h” at a final concentration of 5 µM to the cell suspension, mix well. Incubate at room temperature protected from light and with constant agitation for at least 4 hours (incubation can be extended overnight). 3. Cells Dilution: Dilute cells 3x in assay buffer and incubate as described above for 60 min. 4. Ligands and plates preparation: Prepare serial dilutions of ligands in assay buffer, (2x concentration for agonists, 3x concentration for antagonists). Dispense 50 µL of diluted ligand in a 96-well Optiplate™. Note: Assay can be miniaturized to 384-well and 1536-well formats. 5. Agonist Mode Reading: Using the reader’s automatic injection system, inject 50 µL of cells (i.e. 5 000 cells) per well and immediately record relative light emission for 20-40 seconds. Digitonin at a final concentration of 50 µM in assay buffer is used in control wells to measure the receptor independent cellular calcium response. 6. Antagonist Mode Reading: After 15 minutes of incubation of the cells with the ligand, using the reader’s automatic injection system, inject 50 µL of the reference agonist at a final concentration equivalent to the EC80 and immediately record relative light emission for 20-40 seconds. 7. Data Analysis: The luminescent signal is integrated from second 0 to 20-40, and the integrated value (Area Under the Curve, AUC) is used to draw sigmoidal dose-response curves. Important Notes: Depending on (1) sensitivity of the reader used, (2) plate format used, and (3) assay characteristics wanted, it is possible to load cells at (a) different concentrations of cells and coelenterazine, (b) with different subsequent dilution factors, and (c) using different cell numbers per well. This is part of the validation work when importing an assay to a new reader. For tips and examples on running AequoScreen assays on different readers, please refer to the ® AequoScreen Starter Kit Manual available at www.perkinelmer.com/CellLines. TDS-ES-145-AF-05 Page 4 of 5 ® Materials and Instrumentation The following tables provide the references of compounds and reagents used or recommended for the characterization of the human Chemokine CXCR2 Frozen cells, as well as some advice on how to use these compounds: Table 1. References of compounds used for functional characterization Name IL8 GRO alpha GRO beta SB225005 Provider R&D Systems R&D Systems R&D Systems Calbiochem Cat n° 208-IL 275-GR 276-GB 559405 Working Stock Solution 0.05 mM in PBS-D, 0.1% BSA 0.1 mM in PBS-D, 0.1% BSA 0,01 mM in PBS-D, 0.1% BSA 10 mM in DMSO Table 2. References of cell culture media and assay buffers. Note: The table below lists generic media and additives typically used for PerkinElmer Frozen cells. For product specific media and additives, please refer to the “Recommended Thawing Conditions and Handling of Frozen Cells” section. Name HAM’s F-12 DMEM Advanced DMEM/F12 (serotonin receptors) EMEM EX-CELL DHFR media (DHFR deficient cell lines) FBS FBS dialyzed Calcium and magnesium-free PBS DMEM / HAM’s F12 with HEPES, without phenol red Coelenterazine h Coelenterazine h Digitonin BSA, Protease-free Trypsin-EDTA Sodium Pyruvate L-Glutamine NEAA (non-essential amino acids) Provider Hyclone Hyclone Invitrogen BioWitthaker Sigma Wisent Wisent GIBCO Invitrogen Promega Invitrogen Sigma Sigma Hyclone GIBCO GIBCO GIBCO Cat n° SH30026.02 SH30022.02 12634-010 06-174G C8862 80150 80950 11010 11039-021 S2011 C6780 37006 A-3059 SH30236.02 11360 25030 11140 Please visit our website: www.perkinelmer.com/CellLines for additional information on materials, microplates and instrumentation. This product is not for resale or distribution except by authorized distributors. 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