Cellomics ToxInsight Phospho-ATM Activation Cartridge

Transcription

1 INSTRUCTIONS Cellomics ToxInsight Phospho-ATM Activation Cartridge High-Content Imaging Reagents Number Description Phospho-ATM Activation Cartridge, sufficient materials for 5 96 wells Contents: Phospho-ATM Primary Antibody, 60 μl DyLight 549 Conjugated Goat Anti-Mouse IgG, 72 μl Hoechst Dye, 30 μl Wash Buffer (10X Dulbecco's PBS), 100 ml Wash Buffer II (10X Dulbecco s PBS with 1% Tween -20), 100 ml Permeabilization Buffer (10X Dulbecco s PBS with 1% Triton X-100), 100 ml Blocking Buffer (10X), 85 ml Thin Plate Seal Assembly, 7/pack Storage: Upon receipt immediately store the Phospho-ATM Primary Antibody at -20 C. Store all other components at 4 C. Keep vials containing the fluorescent antibody and Hoechst Dye solutions protected from light. Allow buffers to warm to room temperature before use. See the Solution Preparation section for storage and stability of prepared solutions. Product is shipped with an ice pack. Warning: Completely read these instructions and the accompanying material safety data sheets before using this product. The Cellomics Reagents are not for diagnostic use in humans or animals. Introduction Predictive toxicology of drug candidates remains a major challenge for drug development. Bioethics, efficiency and cost of animal toxicity testing have resulted in a trend toward in vitro toxicity testing earlier in the drug discovery phase to maximize compound attrition as early as possible. Sensitive toxicity assays can provide insight for environmental and industrial safety as well as consumer products such as cosmetics and food additives. The Thermo Scientific Cellomics ToxInsight Platform is a complete solution for cell-based predictive toxicology, comprising of a cellular imaging instrument, software, reagents and other consumables. The ToxInsight Cartridges and associated software protocols are designed specifically for the ToxInsight Instrument. The Cellomics ToxInsight Phospho-ATM Activation Cartridge enables monitoring of DNA damage and cell cycle checkpoint signaling events through ATM phosphorylation (Ser1981) in the nuclei (Figure 1). The cartridge contains a primary monoclonal antibody that detects the phosphorylated form of human ATM, a goat anti-mouse secondary antibody conjugated to Thermo Scientific DyLight 549 Fluorophore, and other reagents and buffers required for immunofluorescence staining for high-content imaging analysis. Ataxia telangiectasia mutated kinase (ATM, 350 kda) is involved in cell cycle check-point signaling and DNA repair. Mutation in the ATM gene leads to ataxia telangiectasia, an autosomal recessive human disease. ATM is auto phosphorylated at Ser1981 upon induction of DNA double-strand breaks, leading to rapid check-point signaling. ATM kinase has several identified targets including H2AX, BRCA1, NBS1, Chk1, Chk2 and p The protocols and example data in these instructions pertain to liver and renal toxicity using HepG2 and HK-2 cells, respectively. Additional protocols and information on other cell types and incubations are available at Pierce Biotechnology PO Box 117 (815)

2 Figure 1. HepG2 cells (A) and HK-2 cells (B) were treated with media (top panels) or 500 µm etoposide (bottom panels) for 3 hours, stained according to the protocol and imaged with the ToxInsight Instrument using the appropriate phspho-atm protocol. Induction of DNA damage by etoposide leads to phosphorylation of ATM. Additional Materials Required Paraformaldehyde (16%) (Thermo Scientific Product No or 28908) 96-well plates (e.g., BD BioCoat Collagen-I coated clear bottom microplates, Product No ) Positive control compound such as etoposide (e.g., Sigma Aldrich Product No. E1383) Fetal bovine serum (e.g., Thermo Scientific HyClone Product No. SH30071) Cell Preparation Information This protocol is optimized for HepG2 cells (ATCC Product No. HB-8065) and HK-2 cells (ATCC Product No. CRL-2190). For routine culture of HepG2 cells, use EMEM medium (e.g., HyClone Product No. SH30244) containing the following supplements: 10% fetal bovine serum, 2 mm L-glutamine, 1 mm sodium pyruvate, 1X non-essential amino acids, 100 units/ml penicillin and 100 μg/ml streptomycin (EMEM complete medium). For routine culture of HK-2 cells, use keratinocyte serum-free medium (e.g., K-SFM; Invitrogen Product No ) supplied with 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml epidermal growth factor (EGF). Also add 2% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin. See the manufacturer s instructions for additional information (K-SFM complete medium). Split cells when they reach 70-80% confluency at a dilution of 1:4 to 1:6 for both HepG2 and HK-2 cells and change media 2-3 times per week. Use cells at a passage number 10. For plating HepG2 cells: Harvest cells by trypsinization (0.25%, w/v) and dilute into EMEM complete medium. Pipette cell suspension vigorously up and down (approximately 20 times) and count cells. If large clumps are still present while counting, pipette suspension another 10 times and count again. Dilute cells to cells/ml in EMEM complete medium. Add 100 μl of the cell suspension per well of a 96-well microplate to achieve 6,000 cells/well. If making multiple microplates, mix cell suspension several times in a reservoir before adding cells to each plate. For plating HK-2 cells: Harvest cells by trypsinization (0.05%, w/v, trypsin-edta solution), dilute into K-SFM complete growth medium. Centrifuge the cell suspension at ~125 g for 5 minutes, discard the supernatant and resuspend the cells in fresh K-SFM medium. After counting, dilute cells to cells/ml in K-SFM complete medium. Add 100 μl of the cell suspension per well of a 96-well microplate to achieve 4,000 cells/well. Incubate cells overnight (18-24 hours) at 37 C in 5% CO 2 before drug treatment. 2

3 Phospho-ATM Activation Cartridge Protocol A. Solution Preparation (per 96-well plate) Positive Control (etoposide) 2X Positive Control Working Solution (1,000 μm) 1X Wash Buffer 1X Wash Buffer II Fixation Solution 1X Blocking Buffer 1X Blocking Buffer with 2% FBS 1X Permeabilization Buffer Primary Antibody Solution Staining Solution Make a 100 mm stock solution of etoposide in DMSO. Store at -20 C until use. Add 60 μl of 100 mm etoposide to 6 ml of warm (37 C) culture medium. Prepare solution just before each assay. Note: This volume is enough for 48 wells in a 96-well plate. Adjust the volume or concentration for different plate setups. Add 20 ml of 10X Wash Buffer to 180 ml of ultrapure water. Store buffer at 4 C for up to Add 4 ml of 10X Wash Buffer II to 36 ml of ultrapure water. Store buffer at 4 C for up to Add 3 ml of 16% paraformaldehyde to 9 ml of 1X Wash Buffer just before use. Warm solution to 37 C before use. Add 3 ml of 10X Blocking Buffer to 27 ml of Wash Buffer. Store buffer at 4 C for up to Add 300 μl of FBS to 15 ml of 1X Blocking Buffer. Prepare solution just before each assay. Add 1.5 ml of 10X Permeabilization Buffer to 13.5 ml of 1X Wash Buffer. Store buffer at 4 C for up to Add 12 μl of Anti-Phospho-ATM Primary Antibody to 6 ml of 1X Blocking Buffer. Prepare solution just before each assay. Add 0.6 μl of Hoechst Dye and 12 μl of the DyLight 549 Goat Anti-Mouse Antibody to 6 ml of 1X Blocking Buffer. Prepare solution just before each assay. B. Procedure 1. Add 100 μl/well of 2X Positive Control Working Solution to the cells (final concentration 500 μm). Add the same volume of media or negative control (e.g., DMSO) to any nontreated wells. Incubate cells for 3 hours at 37 C. 2. Aspirate culture medium and add 100 μl/well of Fixation Solution. Incubate plate in a fume hood at room temperature (RT) for 15 minutes. 3. Aspirate Fixation Solution completely. Wash plate twice with 100 μl/well of 1X Wash Buffer. 4. Aspirate Wash Buffer, add 100 μl/well of 1X Permeabilization Buffer and incubate for 15 minutes at RT. 5. Aspirate Permeabilization Buffer and wash plate twice with 100 μl/well of 1X Wash Buffer. 6. Aspirate Wash Buffer, add 100 μl/well 1X Blocking Buffer with 2% FBS and incubate at RT for 15 minutes. 7. Aspirate Blocking Buffer and add 50 μl/well Primary Antibody Solution. Incubate for 1 hour at RT. 8. Aspirate Primary Antibody Solution and wash with 100 μl/well of 1X Wash Buffer II. 9. Aspirate Wash Buffer II and wash twice with 100 μl/well of 1X Wash Buffer. 10. Aspirate Wash Buffer and add 50 μl/well of Staining Solution containing the goat anti-mouse secondary antibody. Incubate plate at RT for 45 minutes. 11. Aspirate Staining Solution and wash plate twice with 100 μl/well of 1X Wash Buffer II. 12. Aspirate Wash Buffer II and replace with 100 μl/well of 1X Wash Buffer. 13. Seal plate with a provided plate seal and evaluate on the ToxInsight HCS Reader with appropriate protocol and template. Store plates at 4 C when not in use. 3

4 Additional Information A. Fluorescence Absorption/Emission The approximate absorption/emission maxima of the fluorescent dyes are as follows: Hoechst Dye = 350/461 nm DyLight 549 Conjugates = 550/568 nm B. Dose-response Curves HepG2 and HK-2 cells were treated with different concentrations of etoposide for 3 hours as described in the procedure. The fluorescence intensity of phospho-atm was measured and compared to the intensity of negative control (Figure 2). Figure 2. Dose-response curve for etoposide-treated cells. The feature plotted is the %High_CircAvgInten, which is the population of cells with a higher average nuclear intensity of phospho-atm than nontreated control cells. Data represent mean ± SD from three plates (six wells per dose of etoposide per 96-well plate). C. DMSO Tolerance: Assay performance was robust when compounds were added in 1% DMSO. D. Recommendations for Avoiding Cell Loss Treatment with toxic compounds might result in cell loss, which increases data variability. Perform the following procedures to minimize cell loss. Use the lowest speed in liquid handling (i.e., pipetting and aspiration) Centrifuge the microplate before fixation (e.g., ~1,000 g for 5 minutes) Use the collagen net method on cells before drug treatment (see for the protocol) Use automation when possible E. Recommendations for Automation Plating Cells: To improve the uniformity and throughput of plating cells, use a liquid handling system such as Thermo Scientific Multidrop Combi or WellMate Dispensers. Dead Volumes: Every piece of automation instrumentation has a non-recoverable dead volume associated with it. Be aware of these dead volumes, priming volumes and rinsing volumes when calculating your reagent requirements. Nonspecific Binding: Because of the potential of reagent interaction with large surface areas inherent to tubing, syringes and peristaltic pumps, pre-priming with reagents or pre-coating with protein blockers may be warranted. Mixing: Gentle mixing may be required when adding a DMSO-based solution to keep overly concentrated solutions from lying on top of the cell layer. Be careful not to dislodge cells during mixing procedures. 4

5 Cell Washing: Use an automated plate washer designed to gently wash attached cells. Be careful not to dislodge cells during cell washing. Incubation: Minimize the time when plates with live cells are out of a controlled CO 2 environment. For best results, use an automated incubator to deliver plates to a pipetting deck. Exposure: Minimize operator exposure to fixative by some form of containment. Some reagents and compounds are light-sensitive; be aware of these constraints when scaling up for an automated run. Adapting to other plate formats: When using different plate types, adjust cell density and reagent volumes as needed. ToxInsight Protocols The software protocol name is reflective of the Cartridge_Cell Line Tested_Suggested Density_ Suggested Incubation time_plate manufacturer_plate Type. For example: patm_hepg2_6000_3hr_falcon_coli represents HepG2 cells plated at 6,000 cells/well on Falcon Collagen I coated plates treated for 3 hours and using the Phospho-ATM Activation Cartridge) For other cell types and/or conditions, visit References 1. Kastan M.B. and Lim D.S. (2000). The many substrates and functions of ATM. Nature Rev Mol Cell Biol 1: Lavin, M.F., et al. (2005) ATM signaling and genomic stability in response to DNA damage. Mutat Res 569: For Technical Support for the Thermo Scientific Cellomics ToxInsight Cartridges, call Ext (US Toll Free). For more information, visit or call (US Toll Free) or Tween is a registered trademark of ICI Americas. Triton is a registered trademark of Rohm & Haas. This product ( Product ) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product documentation, specifications and/or accompanying package inserts ( Documentation ) and to be free from defects in material and workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than the original purchaser of the Product ( Buyer ). No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular purpose, or non infringement. Buyer s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or refund for the non-conforming Product(s). There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misuse, fault or negligence of or by Buyer, (iii) use of the Products in a manner for which they were not designed, or (iv) improper storage and handling of the Products. Current versions of product instructions are available at Thermo Fisher Scientific Inc. All rights reserved. Unless otherwise indicated, all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. Printed in the USA. 5