TECHNICAL DATA SHEET AequoZen® Caution: For Laboratory Use. A research reagent for research purposes only Human Chemokine CCR6 Receptor, γ-Irradiated Frozen Cells Product No.: ES-139-AF Lot No.: 2462 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): A12 (Cat # ES-000-A12F) Frozen Cells Info: Frozen recombinant, γ−irradiated CHO-K1 cells expressing mitochondrially-targeted Aequorin and the human Chemokine CCR6 receptor. DNA Sequence: Identical to coding sequence of GenBank AY242126.1. Corresponding Protein Sequence: Identical to GenBank P51684.2. 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. MIP-3α (EC50): 0.95 nM Mycoplasma: This cell line tested negative for Mycoplasma. TDS-ES-139-AF-04 Page 1 of 4 Recommended Thawing Procedure Thawing Cells: Using appropriate personal protective equipment, place the frozen aliquot in a 37°C water bath and agitate until its content is thawed completely. Immediately remove from water bath, transfer content to 10 mL assay medium and centrifuge (150 x g, 5 min). Resuspend cell 5 pellet in 33.3 mL (3 x 10 cells/mL) of assay medium for use in the AequoScreen assay (see procedure below). Typical Product Data Emitted Light (RLU) 7000000 MIP-3 alpha 6000000 5000000 MIP-3α 4000000 -10 9.5 x 10 117 3000000 2000000 1000000 0 -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 MIP-3α with at least 16 background and 16 maximal signal points (Z’= 0.98). TDS-ES-139-AF-04 Page 2 of 4 AequoScreen® Assay Procedure (MicroBeta® JET) Assay Buffer: DMEM / HAM’s F12 with HEPES, without phenol red (Invitrogen # 11039-021) + 0.1 % protease-free BSA (filter through 0.22 µm filters). Store at 4°C. Coelenterazine h (500 µM): Solubilize 250 µg of Coelenterazine h (Promega # S2011 or Invitrogen # C6780) in 1227 µL methanol. Store at –20°C in the dark. Digitonin (50 mM): Dissolve 1 g of Digitonin (Sigma # D5628) in 16.27 ml of DMSO. Aliquot and store at -20°C. 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 gentle agitation for at least 4 hours (incubation can be extended overnight). Cell Dilution: Dilute cells 3x in assay buffer and incubate as described above for 60 min. Ligands and plates preparation: Prepare serial dilutions of ligands in assay buffer (2x concentration for agonists, 2x 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. 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 100 µM in assay buffer is used in control wells to measure the receptor independent cellular calcium response. 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. Data Analysis: Sigmoidal dose-response curves are generated using average Luminescent Counts Per Second (LCPS) recorded for 20-40 sec immediately after cells are mixed with the agonist in agonist mode or the EC80 of a reference agonist in antagonist mode. Important Notes: • 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. • 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-139-AF-04 Page 3 of 4 Table 1. References of compounds used for functional characterization Name MIP-3α Provider R&D Systems Cat n° 360-MP Working Stock Solution 0,05 mM in PBS-D, 0.1% BSA Suggested Materials and Instrumentation Please visit our website www.perkinelmer.com/CellLines This product is not for resale or distribution except by authorized distributors. LIMITED WARRANTY: PerkinElmer BioSignal Inc. warrants that, at the time of shipment, the products sold by it are free from defects in material and workmanship and conform to specifications which accompany the product. PerkinElmer BioSignal Inc. makes no other warranty, express or implied with respect to the products, including any warranty of merchantability or fitness for any particular purpose. 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