G1-S Transition (G1ST), Assay Kit

Transcription

1 instructions product code PA55102 G1-S Transition (G1ST), Assay Kit for use in research applications and screens of no more than 1000 compounds per year Warning For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. i PA55102PL Rev-B, 2003

2 Handling Storage Store all components at -70 C. Stability All components are stable for at least 12 weeks when stored under recommended conditions. Expiry The expiry date is stated on the kit label and will normally be at least 12 weeks from the date of despatch. Page finder Handling 2 Components 3 Safety warnings and precautions 3 Other materials required 4 Description 4 Protocol 6 1. Introduction 6 2. Schematic assay protocol 6 3. Schematic assay set up 7 4. Reagent preparation 7 5. Assay protocol 9 6. Assay analysis - principle Assay analysis - IN Cell Analysis System Assay analysis - Confocal microscope system requirements Data and example calculation 20 Additional information 23 Troubleshooting guide 23 Background references 25 Product information 28 Legal 28 2

3 Components Late G1-phase HeLa nuclei: /ml, 25 µl. Asynchronous HeLa cytosol: 0.4 ml. Assay buffer: 5 ml. Premix buffer: 1 ml. Buffered nucleotide mix. Contains less than 1% DTT. Cy5-dUTP: 1.5 nmol. Label, additive to premix buffer. CPK reagent: ~160 units. Creatine phosphokinase buffered solution. Contains 50% Glycerol. Additive to premix buffer. Safety warnings and precautions Warning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals. Handle as a potentially biohazardous material. All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice. 3

4 Other materials required The following materials and equipment are required but not provided with the G1-S Transition Assay Kit. IN Cell Analysis System SYTO13, green fluorescent nucleic acid stain (Molecular Probes, S-7575). Tris-HCl (0.1 M), ph 7.8. Flat field dyes mixture: PBS; Cy 5. monocarboxyl dye (Amersham Biosciences, PA 05101); Fluorescein "reference standard" dye (Molecular Probes, F-1300). Pipettes. Microtiter plate: Packard Black 96-well, # Foil. Plate incubator. Microtiter plate shaker. Confocal microscope SYTO13, green fluorescent nucleic acid stain (Molecular Probes, S-7575). Description The G1ST assay kit utilizes the novel and unique cell-free assay for G1-S phase transition. The initiation of DNA replication is a key regulatory event in the cell cycle, which occurs at the G1-S transition phase. The whole range of signal pathways that induce the cell to enter the division cycle, converge at the G1-S checkpoint. Once replication is initiated, the cell is committed to a full duplication cycle. Therefore, cell cycle regulation (inhibition or stimulation) at the level of initiation of DNA replication is effective and universal, and has the potential to minimize collateral DNA damage, when inhibition is taking place. G1ST kit is based on the principle that nuclei, isolated from human cells, reversibly arrested by mimosine in the late G1-phase of the cell cycle(1), initiate DNA replication in vitro when incubated with cytosolic extracts from asynchronously proliferating or S- phase human cells(2). The kit provides a system for the initiation of semi-conservative DNA replication under somatic cell cycle control(2). G1ST kit presents a unique homogeneous, high-throughput cell-free screening assay that has the capacity to identify inhibitors or stimulators of the initiation of DNA replication on the IN Cell 4

5 Tris-HCl (0.1 M), ph 7.8. Pipettes. Microtiter plate: glass bottom, 96-well. Foil. Plate incubator. Microtiter plate shaker. Software requirements IN Cell Analysis System: Analyzed data is exported in the form of numerical files in ASCII format. These data can be utilized by Microsoft Excel or Microsoft Access or Oracle, or any similar package. Confocal microscope: Image analysis software. ImagePro software is recommended for analysis of images acquired on confocal microscope. Analyzer or on the confocal microscope. The assay is homogeneous, easy to perform and requires straightforward addition of components with minimum dilution steps. Figure 1. Assay principle Late G1- nuclei Test compound 2.5 h incubation, 37 0 C + Cytosol, buffer (nucleotides, including Cy5-dUTP) Initiation of DNA replication Cy5 Late G1- nuclei Incorporation of Cy5-dUTP 5

6 Protocol ❶ Introduction This protocol provides all the information required to use with the G1ST kit to measure the initiation of DNA replication in a cell-free assay on the IN Cell Analyzer or using a confocal microscope. It is recommended that the protocol is read thoroughly before using the kit and that it is followed precisely. Each pack contains sufficient material for 96-wells. Assay incubation time is 2.5 hours followed by 40 minutes incubation with nuclear dye. The fluorescent properties of Cy5 can be adversely affected by exposure to light. We recommend that exposure of Cy5-containing solutions and the microtiter plate during the incubation to all light sources are kept to a minimum. ❷ Schematic assay protocol Add following reagents: Foil plate, Shake on a plate shaker, Incubate plate, 2.5 h, 37 C Assay buffer ± Cytosol ± Test Compound + Nuclei + Premix buffer (with CPK and Cy5-dUTP) Add nuclear dye (e.g. SYTO 13) 6

7 Incubate plate, 20 min, 37 C, then 20 min, RT or ❸ Schematic assay set up Analyze assay (IN Cell Analysis System confocal microscope) Table 1. Reagent amounts per well. Total reaction volume - 50 µl per well. All volumes are in µl. Sample type Assay Cytosol Test Nuclei Premix buffer compound reagent Negative control (G1N cyt) Positive control (G1N + cyt) Compound control (G1N cyt + TC) (35-X) - X 5 10 Compound test (G1N + cyt + TC) (31-X) 4 X 5 10 Where X -variable volume (recommended volume µl); G1N - late G1-phase nuclei; cyt - cytosol; TC - test compound. ❹ Reagent preparation Note: All reagents, except the assay buffer must be allowed to thaw on ice. The assay buffer can be thawed at room temperature and then stored on ice until required. Ensure all components are thoroughly thawed and mixed before use. 7

8 Once diluted, store all components on ice until required. Resuspend nuclei suspension by pipetting up and down, avoid vigorous mixing and generating air bubbles. For nuclei and cytosol: avoid freeze-thaw cycles as they lead to some activity loss. If stock can not be used at once, dispense in suitable aliquots and snap-freeze in liquid nitrogen for further use. Nuclei: Dilute 1/20 in assay buffer for use in the assay, allowing 5 µl of diluted nuclei per well (as shown in Table 1). Cytosol: It is advisable to mix cytosol and assay buffer for simultaneous dispensing, when multiple wells of the same sample type are used (as shown in Table 1). Example: for 10 wells, containing positive control (nuclei with cytosol, G1N + cyt), mix 10 4 µl = 40 µl cytosol and µl = 310 µl assay buffer, to dispense 35 µl of the mixture per well. All amounts are based on quantities per well, suggested in Table 1. This is an example calculation. It is recommended to allow extra volume when the mixture is prepared to ensure sufficient amount of the mixture per well. Premix reagent: Dilute Cy5-dUTP 1/10 in assay buffer: add 13.5 µl of assay buffer to 1.5 µl Cy5-dUTP stock. Per 96-wells: add 50 µl CPK reagent and 12 µl of diluted Cy5-dUTP to 950 µl premix buffer. 10 µl of premix reagent is required per well. Mix well. Protect from light. If less than 96 wells used in the experiment prepare required volume of premix reagent allowing for 10 µl of premix reagent per well. Keep the ratio of components the same. Due to viscosity of premix reagent it is recommended to allow some extra volume when it is prepared to ensure sufficient amount per 8

9 well. If not used at once, store CPK reagent and diluted Cy5-dUTP in aliqouts at -70 C for up to 1 month. This can lead to some activity loss. Repeated freeze-thaw cycles are not recommended. Protect Cy5-dUTP solution from light. SYTO 13: Note: SYTO13 is not provided with the G1-S transition kit. From 5 mm SYT013 stock (Molecular Probes) make up 5 µm by diluting 1/1000 in 0.1M TRIS-HCl (ph 7.8) or any other phosphatefree buffer. 5 µm SYTO 13 solution can be stored up to 1 month at 2 8 C, protected from light. Dilute 5 µm SYTO 13 further 1/40 in 0.1 M TRIS-HCl to make µm SYTO 13 for use in assay. 100 µl of µm SYTO 13 is required per well. ❺ Assay protocol Note: For reagent volumes and sample types refer to Table 1. Ensure correct choice of microtiter plate (see other materials and equipment required). 1. Prepare the reagents as described in reagent preparation. 2. Dispense 35 µl/well of assay buffer into the negative control wells. Dispense (35-X* µl)/well of assay buffer into the compound control wells. 3. Dispense appropriate amounts of assay buffer and cytosol into positive control and compound test wells. Assay buffer and cytosol can be dispensed simultaneously from combined solution: positive control wells (G1N + cyt) have cytosol : assay buffer ratio - 4:31; compound test wells (G1N + cyt + TC) have cytosol : assay buffer ratio 9

10 4:(31-X*), if X* µl per well test compound is to be added). 4. Add X* µl of test compound to corresponding wells (compound control and compound test). 5. Thoroughly but avoiding air bubbles, resuspend diluted nuclei suspension by successive pipetting and add 5 µl nuclei suspension into each well. 6. Add 10 µl premix reagent into each well. 7. Cover the plate with the lid provided, wrap the plate in foil and mix the plate for 1 min on a plate shaker. 8. Incubate the plate at 37 C for 2.5 hours. Half way through the incubation, shake the plate on a plate shaker for sec and return the plate to the incubator. 9. Remove the plate from the incubator and add 100 µl of diluted SYTO13 to each well. Cover the plate with the lid provided, wrap the plate in foil and incubate for 20 minutes at 37 C, then 20 minutes at room temperature. Do not agitate the plate. 10. Analyze on IN Cell Analysis System or confocal microscope. *If compound is to be tested in assay. Note: The order of addition of reagents is very important and the protocol should be followed precisely. ❻ Assay analysis - principle The initiation of DNA proliferation is assessed by incorporation of Cy5-dUTP into nuclei. The nuclear dye SYTO 13 (green) is used to identify nuclei as objects for assessment of the associated intensity of Cy5-dUTP (red). Size filters in the green channel are applied to reject broken nuclei and nuclei clumps. Nuclei are assessed to be positive if the intensity of red signal is 10

11 above the set threshold. Percentage (%) positive nuclei is calculated. ❼ Assay analysis - IN Cell Analysis System requirements For image acquisition and analysis on IN Cell Analyzer refer to the IN Cell Analyzer manual. Object intensity algorithm is recommended to use for data acquisition and real time data analysis on IN Cell Analysis System, where the red and green emissions are collected simultaneously. Flat field: Use FITC and Cy5 dyes solution in PBS for a flat field correction. Recommended amounts per well: 98 µl PBS; 0.4 µl of 150 µμ Cy5 monocarboxyl dye and 0.6 µl of 150 µm FITC dye. Adjust dye concentrations to correlate with the images intensity in the analyzed plate. Recommended settings for G1ST assay analysis on the IN Cell Analyzer. Image Acquisition Start New run. Go to Open new file Take new data based upon an existing run or recipe. 11

12 Select analysis type. Go to Analysis Set Analysis Link Select Analysis Type Object Intensity Analysis. Set up primary parameters. Go to Analysis Primary Analysis Parameters. Select Marker - Green (SYTO 13) and Object - Red (Cy5-dUTP). 12

13 Select Object parameters: The settings shown below are recommended but should be adjusted, if needed (usually the same settings are applied to the same batch of nuclei when assay protocol is followed. Different batches of nuclei may require some parameter settings to be changed). Set background threshold. Set Object Filters, rejecting broken nuclei and nuclei clumps. 13

14 Secondary analysis parameters These can be selected after the plate run has been completed. Go to Analysis Secondary analysis parameters. Apply threshold - choose X standard deviations above background peak, where in the example below X is Note: It is very important to make a correct selection of Secondary threshold. The selection should be based on the intensity profile in a 14

15 negative control (G1N + buf) well. The threshold should be set just above the peak of background intensity. An example of selection of the threshold value is shown below (see arrow in the Signal Intensity Window): 15

16 Then, this threshold is applied to the whole plate (image and intensity histogram, shown below, corresponds to the well containing nuclei and cytosol, G1N + cyt): 16

17 Instrument Settings Go to Instrument Settings panel and set up the instrument settings as shown below. Note: The parameter in the Autofocus Offset window depends on the current instrument focus offset (which can be different from zero). It is recommended to choose this parameter for G1ST assay analysis at 6 µm above Instrument Focus offset. Press: Verify Settings. Go to Plate Setup panel. 17

18 Generate a plate map correspondingly to your experiment. Record Run ID and Plate ID. If needed, write Plate comment. Press: Verify Settings. Go to Run panel. Select % pos (% positive nuclei) in the Legend window. It is recommended to set up the Legend scale parameters to 0, 10 (or 5) and 100. Optional (for scanning observation): open Image, open Population Scatter Plot. Make sure that plate is in the plate hotel. Press: Run. * It is advisable to save Recipe and Analysis Parameters once all parameters, suitable for a particular nuclei batch are selected, and use them for other runs (via commands 'Take new data based upon an existing run or recipe' or 'Import Analysis Parameters'). Otherwise, select a run that can be used as a basis for any new run. Go to Run File. In a 'Select an operating Mode' dialog, choose 'Take a 18

19 new data based upon an existing run or recipe' (see below) 'Open previous run'. Open a file with selected run. All parameters from that run will be loaded. Verify Instrument settings. Update plate map as required and verify Plate settings. Run. Data output Each run on IN Cell Analyzer generates the population data file which is automatically saved in the form of numerical files in ASCII format (as a text-delimited file) and consequently can be opened within Microsoft Excel or Access, or Oracle databases, or other similar package. The resulting file contains a number of parameters, corresponding to each sample (well). '% pos' column represents % positive nuclei, which characterises the level of the initiation of DNA replication in a sample. 19

20 ❽ Assay analysis - Confocal microscope system requirements Image acquisition: Images can be acquired on confocal microscope, following confocal microscope manufactures protocol. Collect Cy5 and SYTO 13 emissions from each frame (field). It is recommended to use objective - 40 and to collect two to four frames per well. Image analysis: Image Pro software is recommended to use for analysis of images (TIFF files), acquired on a confocal microscope. The Macro for G1ST assay analysis in ImagePro is available at the Amersham Biosciences WebSite: ❾ Data and example calculation Typical data obtained with G1ST assay kit. 1. The rate of initiation of DNA replication (late G1-phase nuclei, incubated in buffer without (G1N + buf) and with (G1N + cyt) cytosol, n=8, wells 1A-H and 2A-H correspondingly). Data were obtained on the IN Cell Analysis System, following protocol described in this pack leaflet. Example of calculation The population data file was opened in Microsoft Excel. 20

21 Run ID Date: 06/19/01 Time: 15:29:02 User: ld *** Analysis Parameters Module: Receptor Binding Analysis RCB0.dll Version: Primary Parameters Marker: Green Signal: Red Dilation: 0 Exclude Marker: N Secondary Parameters Positive Thresh: (sdevs above bkgnd) Object Definition Threshold = bkgnd + C Constant C: Discard: Percentile : Close Filter: N Erosion: 0 Filters Bounding Box: [5; 59] Intensity: Off *** End Analysis Parameters Plate Cycle Well Msg Ntot Npass Npos %pos Intensity Std Dev Sig A G1N+buf B G1N+cyt C D z factor 0 E F G H A B C D E F G H %, positive nuclei 0.00 Z factor = 0.71 The mean and standard deviation (SD) of % positive nuclei for wells without and with cytosol were calculated and Z factor(4) was determined, where: Z = 1-3 SD sample + 3 SD control /mean sample - mean control/ 21

22 %, positive nuclei G1N+buf G1N+cyt Z factor = 0.71 n=8 Figure 2. Dose-dependent effect of cdk-inhibitors roscovitine, olomoucine and of the solvent DMSO on the initiation of DNA proliferation. Results were obtained on the IN Cell Analysis System following protocol as described in the pack leaflet. % positive nuclei Roscovitine Olomoucine Control (DMSO) Inhibitors, mm 22

23 Additional information Troubleshooting guide Problems Possible causes and remedies ❶ Low % of initiated nuclei in positive control (nuclei incubated with cytosol) ❷ High % of initiated nuclei in negative control ❸ Image is out of focus Possible cause 1.1: Incorrect selection of analysis parameters. Remedy 1.1: Check if your primary parameters are correct and suitable for a nuclei batch that is currently in use. Check if your secondary threshold selection is correct and suitable for a nuclei batch currently in use. Possible cause 1.2: The assay reagents were not mixed properly. Remedy 1.2: Ensure reagents are mixed thoroughly. Possible cause 1 3: Incorrect conditions of assay incubation. Remedy 1.3: Check incubation time and temperature. Possible cause1.4: Reagents were not stored properly or they are out of date. Remedy 1.4: Repeat assay with fresh reagents. Possible cause 1.5: Nuclei or cytosol were defrosted previously, but were not re-frozen properly. Remedy 1.5: If not used at once, snap-freeze nuclei and cytosol in liquid nitrogen and store at 70 C for further use. Possible cause 2.1: See 1.1. Remedy 2.1: See 1.1. Possible cause 3.1: Autofocus offset is chosen incorrectly. Remedy 3.1: Change autofocus offset. Possible cause 3.2: Plate was agitated. Remedy 3.2: Allow nuclei to settle before reading the plate. 23

24 Problems ❹ Many broken nuclei are seen ❺ Excessive clumping of nuclei ❻ Precipitants in reaction solution Possible causes an remedies Possible cause 1.1: Nuclei were resuspended too vigorously. Remedy 1.1: Take care when pipetting and resuspending nuclei. Possible cause 5.1: Nuclei were not resuspended sufficiently. Remedy 5.1: Resuspend nuclei well (but not too vigorously) before dispensing. Possible cause 6.1: Precipitation in buffers Remedy 6.1: Filter assay buffer and Tris buffer via 0.22 µm filter. Possible cause 6.2: Precipitation in cytosol. Remedy 6.2: Spin cytosol briefly (for 10 sec) at 1000 g before use. 24

25 Background references 1. Krude, T., Exp. Cell Res. 247, (1999). 2. Krude, T., J. Biol. Chem. 275(18), (2000). 3. Leatherwood J., Curr. Opin. Cell Biol 10, (1998). 4. Zhang, L. et al., J. Biomol. Screen. 4, (1999). 25

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28 Legal Cy ia a are trademark of Amersham Biosciences Amersham and Amersham Biosciences are trademarks of Amersham plc Microsoft is a trademark of Microsoft Corporation Oracle is a trademark of Oracle Corporation Use of this product is limited to research applications and screens of no more than 1000 compounds per year. A license is required to screen greater than 1000 compounds per year. Some of these products may only be available to collaborators and customers within certain of our technology access programmes All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences Goup which supplies them. A copy of these terms and conditions is available on request. Amersham Biosciences UK Limited 2003 All rights reserved Product information Product name code G1-S transition assay (G1ST), 100-well kit PA Related products Cy5-dUTP 25nmol PA55022 G1-S transition assay (G1ST), 1000-well kit* PA55002 G1-S transition assay (G1ST), well kit* PA55202 *Prior to ordering of these products a licence for high-throughput screening (more than 1000 compounds per year) will be required. Contact your local representative for full details. This kit is manufactured under license from Cancer Research Campaign Technology Limited. The technology is covered by patents US 6,107,042, AU 9,733,509 and other patents pending. The Cyanine dye in this product is manufactured by Amersham Biosciences, under an exclusive license from Carnegie Mellon University, and is covered by US patent number 5,268,486 and other patents pending. Use of products for commercial purposes is strictly forbidden without permission from Amersham Biosciences Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK Amersham Biosciences AB SE Uppsala Sweden Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D Freiburg Germany 28