Cytiva Cell Health Assay for HCA

Transcription

1 GE Healthcare Life Sciences Cytiva Cell Health Assay for HCA Product booklet Code: wells wells

2 Page finder 1. Legal 4 2. Handling Safety warnings and precautions Storage Expiry 5 3. Components of the assay system 6 4. Description 7 5. Additional equipment and reagents required Critical Factors System protocols Suggested experimental setup Setup for 96-well plates Reagent preparation well assay protocol Setup for 384-well plates Reagent preparation Assay protocol Image Capture Image Analysis Cell health assay kit- 4-Reagent assay set up Overview Analysis workflow Cell health assay kit - set up for 3-Reagent and 2-Reagent assays Assay 1-3-Reagent assay Overview 36 2

3 Analysis workflow Assay 2-2-Reagent viability assay Overview Analysis workflow Image Analysis IN Cell Investigator Data Handling Troubleshooting guide References Related products 47 3

4 1. Legal GE, imagination at work and GE monogram are trademarks of General Electric Company. Cytiva is a trademark of GE Healthcare Companies. Spotfire is a trademark of Tibco Software Inc. For research use only. Not to be used for any therapeutic or diagnostic applications. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information General Electric Company All rights reserved. First published September GE Healthcare UK Limited Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK 4

5 2. Handling 2.1. 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. 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 Storage Store Reagent A and Reagent B at 2-8 C. Store Reagent C and Reagent D at -20 C Expiry The expiry date is stated on the package and will be at least 6 months from the date of despatch. 5

6 3. Components of the assay system The pack contains the following assay components: sufficient material for 2 96-well or well plate. Reagent A and Reagent B must be stored at 2 8 C, Reagent C and Reagent D must be stored at -20 C. Reagent Excitation/ Emission peaks (nm) Quantity provided in kit Reagent function Report A 355/ µl Membrane permeant nuclear stain B 540/ µl Membrane impermeant nuclear stain C 548/ µl Membrane permeant indicator for mitochondrial morphology and mitochondrial membrane potential D 488/ µl Membrane permeant calcium indicator Cell count, Nuclear morphology and DNA content Viability Mitochondrial health Calcium levels Table 1. Assay components including peak excitation/emission wavelengths (nm) for each dye reagent and description of function and cellular measure reported. 6

7 4. Description The Cytiva Cell Health assay kit provides a high content analysis (HCA) assay that has been designed to monitor multiple toxicity indicators and cellular parameters to achieve greater sensitivity and information compared with single endpoint toxicity assays. The Cytiva Cell Health assay kit has been designed to maximise the power of cell models such as Cytiva Cardiomyocytes in applications related to compound toxicity prediction. Relatively simple cell models based on immortalized cell lines coupled with single endpoint cytotoxicity assays are not always able to predict organ specific toxicities 1. The predictivity of in vitro models can be greatly enhanced by choosing cell models that better reflect the in vivo tissue and by monitoring multiple parameters to achieve greater sensitivity and information compared with single endpoint assays. In preparation for the assay, cells are cultured in the presence of test substances in a 96- or 384-well microplate at 37 C. Following an extended incubation period, cells are loaded with the dye components from the kit for 1 hour before an image capture step using either a HCA platform or an automated fluorescence microscope. Acquired images should then be analyzed using automated image analysis software. In one set of reagents (one vial each of Reagent A, B, C and D) there is a sufficient material to perform the assay on a minimum of 96 wells. For experiments performed over extended time courses greater than 24 hours it is recommended that outer edge wells of cell culture plates not be used for evaluation of test article toxicity due to the different growth conditions in these wells. Figure 1 features an example experiment whereby only the central 60 wells of a 96-well plate are used to evaluate test article toxicity. Users are encouraged to perform the experiment in accordance with their own preferred experimental setup. 7

8 Figure 1. The Cell Health Assay can be performed with all reagents combined and imaged together as a 4-Reagent assay which is the recommended set up for the assay. Due to the similarity of the excitation/emission spectra of Reagents B and C, they should be imaged simultaneously using the same optical settings. Therefore on systems that lack the capability to segment images for the purpose of applying a live/dead filter step, there is the option of performing the procedure as two separate assays incorporating a live/ dead 2-Reagent assay and a 3-Reagent assay for monitoring intracellular calcium and mitochondria changes. Double the amount of Reagent A has been provided since it is required for both the 3- and 2- Reagent assay setups. 8

9 Figure 2. Images of Cytiva Cardiomyocytes seeded at cells per well taken using an IN Cell Analyzer 2000 with a 40 objective demonstrating a range of cellular responses upon treatment with compounds. Treatment with FCCP leads to complete disruption of mitochondrial staining (Reagent C red mitochondrial staining diminished compare with untreated cells with representative mitochondrial staining highlighted with yellow arrows). Treatment with 0.025% Triton X-100 leads to membrane disruption and staining of nuclei with both Reagent A and B (Nuclei of viable cells are only stained with Reagent A whilst membrane disrupted cells stain with both Reagent A and B resulting in magenta appearance of nuclei). Treatment with Amiodarone for 72 hours leads to changes in mitochondrial morphology (Reagent C red staining no longer uniform i.e. often truncated or aggregated) and an increase in Calcium (Reagent D green staining). 9

10 5. Additional equipment and reagents required Pipettes or pipetting equipment with disposable tips. Epifluorescence microscope with automated multiwell plate compatible stage or High Content imaging platform with 40 and/or 20 objective. Excitation/Emission filters suitable for the excitation and emission peaks of the dye components shown in Table 1. Image analysis software capable of nuclear and mitochondrial segmentation with sub-population filtering capabilities (to define live and dead cell sub-populations and further analyze live cell population). Inverted cell culture microscope for routine cell culture to assess suitability of cell assay plates for HCA. Haemocytometer or other cell counting methodology. Tissue culture grade 96 or 384-well microplate suitable for imaging applications, seeded with cultured cells of choice. Test substances Compounds, toxins or other substance to be tested. Suggested control compounds for the assay: Ionomycin - (Sigma-Aldrich Cat# i0634-5mg) FCCP - (Sigma-Aldrich Cat# c mg) Triton X-100 (Sigma-Aldrich Cat# ml) Tissue culture incubator. Tissue culture medium. 10

11 6. Critical Factors The following points are critical: Allow all reagents to reach room temperature before assay. Briefly spin tubes before opening to ensure dye is at bottom of tube. Once dyes are added to media, mix well before adding to assay plate. Once dyes are added to assay medium, transfer to the cell assay plate should be conducted immediately for optimum dye performance. When using an automated imaging system, ensure that there is sufficient storage space for image files before initiating the scan. After the scan, check the destination folder to ensure that images have been successfully stored along with any associated files. The plate scanning process should not exceed 90 minutes, since exceeding this limit can lead to time-dependent artefacts across the plate. 11

12 7. System protocols 7.1. Suggested experimental setup A typical workflow would be to prepare a 3 Compound Plate and then transfer compounds from this compound plate to the Cell Assay Plate, which contains the cultured cells. Incubation time will depend on the nature of the test substances and cell type employed, and typically ranges from a few hours to up to 3 days. Users should aim to perform the assay using an 8-10 dose point range from sub-millimolar to sub-micromolar levels; for example a dose range from 300 µm down to 0.03 µm should capture most biologically relevant responses with most compound classes. Results obtained in wells along plate edges may vary 2. For this reason, although enough reagent is provided to use in all wells of a 96 - or 384-well plate, the outside wells are often avoided (as in the plate map shown in sections 7.2 and 7.3). However, these wells are typically seeded and used for functional dye control treatments on the day of the assay (see sections and 7.3.2). Note: Cells should be seeded into wells at a density that will result in a well coverage of 85-95% confluence on the day of analysis. For example, HepG2 cells are typically seeded at 4000 cells per well of a 96-well plate which results in near confluent cell coverage after 4 days in culture. Initial cell seeding densities that result in overly confluent wells should be avoided as this can complicate the image analysis step, particularly in HCA platforms that lack confocal or other optical sectioning capability. 12

13 7.2. Setup for 96-well plates To 100 µl of media in a well, add 50 µl of test substance from a compound plate prepared at 3-fold the final intended compound concentrations. For example, addition of 50 µl of 900 µm compound to a well containing 100 µl of media yields 300 µm final concentration of the test substance. Outside wells can be seeded with cells and used for dye control treatments (see section 7.2.2) on the day of assay. A B C D E F G H Vehicle Vehicle 0.03 µm 0.03 µm 0.09 µm 0.09 µm Vehicle 0.03 µm 0.09 µm 0.30 µm 0.30 µm 0.30 µm 0.95 µm 3.00 µm 9.5 µm 30 µm 95 µm 300 µm 0.95 µm 3.00 µm 9.5 µm 30 µm 95 µm 300 µm 0.95 µm 3.00 µm 9.5 µm 30 µm 95 µm 300 µm Compound 1 Compound 2 Compound 3 Figure 3. Example Cell Assay 96-Well Plate layout. The 96-well plate layout in Figure 3 shows a 9 point dose range for 3 compounds from 300 µm to 0.03 µm plus a vehicle-only control. The serial dilution for the dose range is 3.16 and can be achieved by addition of 100 µl of compound into of diluent media. Thus for the experimental setup above, the following workflow would be appropriate: 13

14 A. Prepare 30 mm stock solution of each compound to be tested in sterile vehicle solvent of choice such as DMSO, media, methanol or other suitable solvent. B. Perform a 33.3 dilution of dissolved compound into media by adding 30 µl of compound into 970 µl of culture media to give the top 900 µm compound concentration. Note: This is the 3 concentration for the highest dose for the assay plate. C. To prepare a 3 Compound Plate, for each compound transfer duplicate 320 µl amounts of the 900 µm compound concentration into column 11 of a clean sterile 96 well plate of suitable volume capacity A 320 µl into column 11 B C D E F G H Compound 1 Compound 2 Compound 3 Figure 4. Example 3 Compound 96 Well Plate layout. To begin the serial dilution, compound (or other test substance) is added to column 11 at 3 times the highest intended final concentration on the assay plate. D. Into columns 2 through to 10 dispense of media containing 3% (v/v) of the appropriate vehicle into each well. This ensures that, following addition of compound to the cell assay plate, the concentration of vehicle is constant across the plate. 900 µm 900 µm 900 µm 14

15 A B C D E F G H Compound 1 Compound 2 Compound 3 Figure 5. Serial dilution of the 3 Compound 96 Well Plate. of media containing 3% (v/v) of the appropriate vehicle is added to wells in columns 2 through to 10. Serial dilutions are then made from right to left across the plate by transferring 100 µl of compound each time. E. Using a multi-channel pipette, perform serial dilution from right to left across the plate by transferring 100 µl of the compound into of media in the neighbouring well (i.e. for the first step, 100 µl of well B11 would be transferred into well B10). Mix thoroughly by 4 repeated pipetting strokes and continue with the serial dilution until column 3 of the plate. F. Once the serial dilution is completed the cells can be treated by transfer of 50 µl from the 3 Compound Plate into the Cell Assay Plate containing 100 µl media per well. G. To avoid cross contamination it is advisable to use a multichannel pipette and to add from column 2 across to column

16 It is recommended that the outside wells be seeded with cells but not be used for testing compound due to the increased evaporation rates at the edge of the plate. These wells can be used for dye loading (see section 7.2.2) and function controls on the day of assay Reagent preparation A. Remove dye reagents from storage and equilibrate to room temperature prior to use. For a 96-well plate, one tube of each reagent should be used. Note: Reagents C and D are supplied as DMSO solutions and can be warmed in a 37 C water bath to expedite thawing. B. Once all reagents have thawed, briefly spin tubes in microcentrifuge to ensure contents are at the bottom of the tubes. C. Warm cell growth media by placing in a 37 C water bath. D. For one 96-well plate, add assay components as indicated in Table 2 to a sterile tube and mix well. Note: Freeze-thaw of stock reagents is not recommended. Do not re-use reagents once prepared. 16

17 96-Well Plate 4 Dye Mix Item Volume added 4-Reagent Assay 3-Reagent Assay 2-Reagent Assay Growth media 6 ml Reagent A 25 µl Reagent B 25 µl Reagent C 25 µl Reagent D 25 µl Table 2. 4 Dye mix for a 96-well cell assay plate. Prepare a sterile tube containing the indicated volumes of assay components. Tick marks indicate which reagents should be added depending on which assay format is being prepared i.e. 4 / 3 / 2 Reagent assay well assay protocol A. Decant the dye mix into a sterile reagent reservoir and dispense 50 µl into each treated well of a 96-well cell assay plate. Return the plate to the incubator for 1 hour before proceeding to image capture step. If the assay is being performed as the 2-Reagent and 3-Reagent assay format, then the plates should be loaded with dye at a time interval that would allow both plates to be scanned consecutively. Suggested Dye Controls: To set up dye controls for the assay plate, treat designated control wells with the following compounds for 20 minutes before loading cells with the dye mix: Untreated For comparison with vehicle an untreated control can be included by adding 50 µl of media to a well. Ionomycin Calcium ionophore that results in significant increase in intracellular calcium. Titration of this reagent is recommended to optimize the concentration for each cell line, although 20 µm has been shown to be a suitable concentration for several common cell lines. 17

18 FCCP (Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone) Protonophore that leads to depolarization of mitochondria resulting in a loss of mitochondrial staining (as monitored using reagent C). A concentration of 10 µm has been shown to be a suitable concentration for several common cell lines. Triton X-100 Non-ionic surfactant that solubilizes the cell membrane and is used as a control for staining of cells with compromised membrane integrity and is useful in setting a threshold for software filtering of dead cells from subsequent image analysis. A concentration of 0.025% has been shown to be a suitable concentration for several common cell lines. A B C D E F G H Untreated Ionomycin FCCP 10 µm Triton X100 Untreated 20µM (0.025%) Untreated Ionomycin FCCP 10 µm Triton X100 20µM (0.025%) Untreated Compound 1 Compound 2 Compound 3 Figure 6. Suggested placement of dye controls on cell assay plate. 18

19 7.3. Setup for 384-well plates For a 384 well plate, both sets of reagent tubes from the kit should be used. To 40 µl of media in a well, add 20 µl of test substance from a compound plate prepared at 3-fold the final intended compound concentrations. For example addition of 20 µl of 900 µm compound to a well containing 40 µl of media yields 300 µm final concentration of the test substance. Outside wells can be seeded with cells and used for dye control treatments (see section 7.3.2) on the day of assay. Figure 7. Example Cell Assay 384 Well Plate layout. 19

20 The 384-well plate layout in Figure 7 shows a 9 point dose range for 8 compounds from 300 µm to 0.3 µm plus a vehicle-only control. The serial dilution for the dose range is 3.16 and can be achieved by addition of 25 µl of compound into 54 µl of diluent media. Thus for the experimental setup above, the following workflow would be appropriate: A. Prepare 30 mm stock solution of each compound to be tested in sterile vehicle solvent of choice such as DMSO, media, methanol or other suitable solvent. B. Perform a 33.3 dilution of dissolved compound into media by adding 9 µl of compound into 291 µl of culture media to give the top 900 µm compound concentration. Note: This is the 3 concentration for the highest dose for the assay plate. C. To prepare a 3 Compound Plate, for each compound transfer 80 µl amounts into the indicated wells (columns 12 and 22) of the 900 µm compound concentration into a clean sterile 384 well plate of suitable volume capacity. 20

21 Figure 8. Example 3 Compound 384 Well Plate layout. To begin the serial dilution, compound (or other test substance) is added to columns 12 and 22 at 3 times the highest intended final concentration on the assay plate. D. Into columns 3 through to 11 and 13 through to 21 dispense 54 µl of media containing 3% (v/v) of the appropriate vehicle into each well. This ensures that, following addition of compound to the cell assay plate, the concentration of vehicle is constant across the plate. 21

22 Figure 9. Serial dilution of the 3 Compound 384 Well Plate. 54 µl of media containing 3% (v/v) of the appropriate vehicle is added to wells in columns 3 through 11 and 13 through to 21. Serial dilutions are then made from right to left across the plate by transferring 25 µl of compound each time. E. Using a multi-channel pipette, perform serial dilution from right to left across the plate by transferring 25 µl of the compound into 54 µl of media in the neighbouring well (i.e. for the first step, 25 µl of well C12 would be transferred into well C11). Mix thoroughly by 4 repeated pipetting strokes and continue with the serial dilution until column 4 of the plate. F. Once the serial dilution is completed the cells can be treated by transfer of 20 µl from the 3 Compound Plate into the Cell Assay Plate containing 40 µl media per well. G. To avoid cross contamination it is advisable to use a multichannel pipette and to add from left to right across the plate and changing pipette tips where there is a change in compound. 22

23 It is recommended that the outside wells be seeded with cells but not be used for testing compound due to the increased evaporation rates at the edge of the plate. These wells can be used for dye loading (see section 7.3.2) and function controls on the day of assay Reagent preparation A. Remove dye reagents from storage and equilibrate to room temperature prior to use. For a 384-well plate, two tubes should be used. Note: Reagents C and D are supplied as DMSO solutions and can be warmed in a 37 C water bath to expedite thawing. B. Once all reagents have thawed, briefly spin tubes in microcentrifuge to ensure contents are at the bottom of the tubes. C. Warm cell growth media by placing in a 37 C water bath. D. For one 384-well plate add assay components as indicated in Table 3 to a sterile tube and mix well. For a single 384 well plate, 12 ml of the 4 dye mix can be prepared by using both sets of reagent tubes from the kit. See Table 3. 23

24 384 Well Plate 4 Dye Mix Item Volume added 4-Reagent Assay 3-Reagent Assay 2-Reagent Assay Growth media 12 ml Reagent A 50 µl Reagent B 50 µl Reagent C 50 µl Reagent D 50 µl Table 3. 4 Dye mix for a 384-well cell assay plate. Prepare a sterile tube containing the indicated volumes of assay components. Tick marks indicate which reagents should be added depending on which assay format is being prepared (i.e. 4 / 3 / 2 Reagent assay.) Assay protocol A. Decant the dye mix into a sterile reagent reservoir and dispense 20 µl into each treated well of a 384-well cell assay plate. B. Return the plate to the incubator for 1 hour before proceeding to image capture step. If the assay is being performed as the 2-Reagent and 3-Reagent assay format, then the plates should be loaded with dye at a time interval that would allow both plates to be scanned consecutively. Suggested Dye Controls: To set up dye controls for the assay plate, treat designated control wells with the following compounds for 20 minutes before loading cells with the dye mix: Untreated For comparison with vehicle an untreated control can be included by adding 50 µl of media to a well. Ionomycin Calcium ionophore that results in significant increase in intracellular calcium. Titration of this reagent is recommended to optimize the concentration for each cell line, although 20 µm has been shown to be a suitable concentration for several common cell lines. 24

25 FCCP (Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone) Protonophore that leads to depolarisation of mitochondria resulting in a loss of mitochondrial staining (as monitored using reagent C). A concentration of 10 µm has been shown to be a suitable concentration for several common cell lines. Triton X-100 Non-ionic surfactant that solubilises the cell membrane and is used as a control for staining of cells with compromised membrane integrity and is useful in setting a threshold for software filtering of dead cells from subsequent image analysis. A concentration of 0.025% has been shown to be a suitable concentration for several common cell lines. Triton FCCP Ionomycin Untreated Untreated Ionomycin FCCP Triton Figure 10. Suggested placement of dye controls on cell assay plate. 25

26 8. Image Capture Recommended wavelength and bandwidth for image capture Reagent Excitation / Bandwidth (nm) Emission / Bandwidth (nm) Output image A 350 ± ± 25 Blue Channel B 579 ± ± 20 Orange Channel C 579 ± ± 20 Orange Channel D 490 ± ± 18 Green Channel Table 4 Suggested optical setup for image capture step. Note: Ensure that there is sufficient storage space available for the image files. The amount of storage space required will differ depending on the number of wells, fields per well and the camera used. Users are advised to take this into account when assessing the required storage space needed. However, as a guide an IN Cell 2000 Analyzer fitted with a large camera and taking 4 fields of view for a total of 80 wells will require approximately 8 GB of storage space. The Cell Health Assay kit has been optimized for use with HCA and automated microscopes able to image using Excitation/Emission optics compatible with the spectral ranges detailed for each of the reagents in Table 4. Due to the similarity of the excitation/emission spectra of Reagents B and C, they should be imaged simultaneously using the same optical settings. Since the assay is performed on live cells it is recommended that the instrument be capable of maintaining the cell assay plate at the conditions used for culturing the cells (37 C, 5% CO2). Changes 26

27 in mitochondrial morphology are one of the earliest indications of cellular stress. To capture such changes in sufficient detail, it is recommended that images be acquired using a 40 objective lens³. For each channel, the exposure time and any focus offset should be optimized according to the following sequence: Reagent A (Nuclear): Select an untreated or vehicle-treated well with no or few dead cells, perform focus and adjust exposure time to a level that results in 40% saturation +/- 10 %. Reagent B (Viability): No optimization step required but viewing of Triton X-100 treated wells should reveal similar nuclear staining pattern as for Reagent A. Reagent C (Mitochondria): Select an untreated or vehicletreated well with no or few dead cells, perform focus and adjust exposure time to a level that results in 40% saturation +/- 10 %. Reagent D (Calcium): Select an untreated or vehicle-treated well with no or few dead cells, perform focus and adjust exposure time to a level that results in 10% saturation +/- 5%. Alternatively, select a well treated with ionomycin and select an exposure time that results in almost total saturation of the camera. Once exposure times have been optimized, select an untreated well and set Z offsets according to best image focus for Reagents A, C and D (Reagent B and Reagent C are imaged together). 27

28 Once image capture is optimized, set instrument to image a minimum of 250 cells per well (untreated or vehicle-treated). For example a 96-well plate of 80% confluence will have approximately 80 cells per field if captured using a 40 objective on an IN Cell Analyzer 2000 fitted with the large camera. Thus it would be recommended to capture a minimum of 4 fields (4 80 = 320 cells) per well. Note: It is advized that the plate scanning process should not exceed 90 minutes as this can lead to time dependent effects across the plate. Once the scan is complete, check that images and any associated files are present in appropriate storage location and then proceed to image analysis. Imaging on IN Cell Analyzer platforms Users of IN Cell Analyzer 1000, IN Cell Analyzer 2000 and IN Cell Analyzer 6000 can find example acquisition protocols specific for this assay at 28

29 9. Image Analysis Cytiva Cell Health for HCA kit allows the assessment of cell number (Reagent A staining), nuclear morphological characteristics (Reagent A staining); cell viability (Reagent B staining), mitochondrial status (Reagent C staining) and cellular calcium levels (Reagent D staining). As demonstrated in Section 4, Cytiva Cell Health kit assay can be set up as a 4-Reagent assay or as a combination of 2- and 3-Reagent assays. Imaging guidelines below are given for each option of the assay formats Cell health assay kit - 4-Reagent assay set up (Reagents A,B,C and D are added into reagent mix at the same time) Overview Based on fluorescent properties of the kit components and imaging capabilities of the majority of fluorescent imaging devices, images from this assay will be collected into 3 channels: 29

30 Reagent Excitation/ Emission peaks (nm) Output image Objects identified Report A 355/465 Blue Channel All Nuclei Cell count Nuclear morphology and DNA content B 540/608 Orange Channel C 548/573 Orange Channel Dead cells nuclei Mitochondria % Viability Mitochondria quantity and formation D 488/515 Green Channel Cellular Calcium Calcium levels Table 5. Description of assay-reagents including spectral properties, output channels, organelles identified and output measures (4-Reagent assay set up) Analysis workflow Images are analyzed by first defining cells, based on the nuclear stain (Reagent A), followed by removal of dead cells from subsequent analysis on the basis of the nuclear staining by Reagent B. The remaining viable cell population can then be analyzed for indicators of cell health such nuclear morphology (Reagent A), intracellular calcium levels (Reagent D) and mitochondrial form and mass (Reagent C). In a 4-Reagent assay set-up, images from Reagent B and Reagent C are collected in the same channel (Orange Channel), which should be taken into account when mitochondria are segmented. Carry out the following steps: 1. Identify objects of interest in the images Identify (segment) nuclei in the Blue Channel image (Reagent A) Depending on the image quality, a pre-processing step (such as shading removal, de-noising, etc.) may be 30

31 required to prepare images for better nuclei identification (segmentation) Apply a segmentation method to separate nuclei from background and each other, so that the segmentation result corresponds as closely as possible with the objects observed in the original image, as illustrated in the example shown in Figure 11 (adjust image visuals to make nuclei clearly visible in the image). Figure 11. A Original image of Reagent A staining. B Result of nuclear segmentation (blue - nuclei mask) If required, use post-processing steps, such as erosion/ dilation, fill holes, sieve, and clump breaking to improve segmentation results (improve position of outlines and separation between closely located nuclei; remove debris, etc.) Generate a Cytoplasmic sampling region Using tools available in your image analysis software package, define a cytoplasmic sampling region. For example, based on the defined nuclear mask, a collar (a ring around nuclei, as demonstrated in Figure 12) can be generated to sample intensity values in the cytoplasmic area immediately adjacent to the nucleus - a cytoplasmic sampling area. 31

32 Figure 12. A Nuclei image (Reagent A) - a collar sampling region (yellow outline) was generated on the basis of the nuclear mask (blue outline); B Fused image of all 4 reagents (3 channels). Cytoplasmic sampling region yellow outline Identify (segment) mitochondria in the Orange channel image (Reagent C) Apply a segmentation method to separate mitochondria (presented in the image as regions of granular or threadlike staining within cytoplasm) from background or any non-mitochondrial objects such as nuclei Due to their similar fluorescent properties, Reagent C and Reagent B are imaged using the same excitation and emission conditions. Thus the image acquired in the Orange channel will capture both mitochondrial and nuclear staining in this assay set up*. However, cellular locations of Reagent C and Reagent B staining are spatially separated with differing morphological characteristics and, in the majority of cases, differing intensity levels. These differences allow extraction of measurements specific for each marker, from the same image. * see 3- and 2-Reagent assay set up (section 9.3) for alternative assay protocol, resulting in the separate collection of images from Reagents B and C. 32

33 To achieve this, it is important to ensure that mitochondrial segmentation accurately identifies mitochondria only, excluding Reagent B staining of dead cell nuclei, as shown in Figure 13. This can be accomplished with addition of post-processing steps following the initial segmentation procedure. Depending on the segmentation method used, one or more filters based on size or intensity may beused. For example, a size filter can effectively exclude objects larger than mitochondria (e.g. nuclei). Since reagent B signal is typically of higher intensity than Reagent C signal, an intensity filter may also be of value in excluding the dead cell nuclei. Figure 13. H9C2 cells 1 hour after addition of the 4-dye mix. A Fused image, capturing staining of cell nuclei (blue), cell mitochondria (thread-like morphology, red), and dead-cell nuclei (red). Intracellular calcium signal (green) in one of the cells is evident as intense orange/magenta (due to colour overlap from multiple channels comprising the fused image). B Orange channel image only, revealing staining of both mitochondria and dead cell nuclei; C Live-cell mitochondrial segmentation (magenta mask); dead-cell nuclei have been effectively excluded from analysis (no mask). 33

34 2. Define live-cell and dead-cell sub-populations Using the nuclear mask generated in step 1.1, measure Average Intensity of signal from Reagent B (Orange channel), i.e. intensity of Reagent B signal will only be measured in the region of nuclei Reagent B selectively stains dead cells (cells with compromised plasma membrane) only. Set up a threshold for reagent B Average Intensity to separate live and dead cells as shown in Figure 14. Figure 14. Identification of dead cells. A Nuclei staining (Blue Channel); B Image of dead cells nuclei staining and mitochondria staining (Orange channel); C A threshold is set for nuclei average intensity in the Orange image (dead cell staining) to separate dead and live cells; D Fused image of 4-Reagent assay set up (blue nuclei stain; green calcium; red mitochondria and dead nuclei staining). Data table (left) shows Average nuclei intensity values collected from the image in the Orange channel (dead nuclear staining). Highlighted values correspond to highlighted (with yellow outline) nuclei, one of which is a nuclei of dead cell (Average intensity value is above the set threshold (845 vs. 450). 34

35 2.3. Report number of all cells and percentages of live and dead cells as an indication of cell viability. 3. Collect cell health information about live cells. Excluding dead cells from analysis, apply the following measurement to the live-cell sub-population: 3.1. Nuclear morphology and intensity of staining Use nuclear mask to measure morphological parameters from nuclei (e.g. Nuclear area and Form factor) and total intensity of nuclear staining as an indicator of cellular DNA content (Blue channel) Calcium status Use Cell sampling region to measure intensity of calcium signal (Green channel) as an indicator of intracellular calcium levels Mitochondrial health Using mitochondria mask, collect measures from Reagent C (Orange Channel) image, such as Total organelle area, as an indicator of mitochondrial mass; organelle form factor, as an indication of mitochondrial morphology Cell health assay kit - set-up for 3-Reagent and 2-Reagent assays With this optional format, two separate assays are configured to be run independently: Assay 1 - Reagents A, C and D are combined in the reagent mix. Assay 2 - Reagents A and B are combined in the reagent mix. 35

36 9.3. Assay 1 3-Reagent assay Overview Based on the fluorescent properties of the kit components and the imaging capabilities of the majority of fluorescence imaging devices, images from this assay will be collected into 3 channels. Reagent Excitation/ Emission peaks (nm) Output image Objects identified Report A 355/465 Blue Channel All Nuclei Cell count Nuclear morphology and DNA content C 548/573 Orange Channel Mitochondria Mitochondria quantity and formation D 488/515 Green Channel Cellular Calcium Calcium levels Table 6. Description of assay reagents including spectral properties, output channels, organelles identified and output measures (3-Reagent assay set up) Analysis workflow Images are analyzed by defining cells, based on the nuclear stain (Reagent A). Mitochondria are segmented based on Reagent C staining. Cells are then analyzed for indicators of cell health such as nuclear morphology (Reagent A), intracellular calcium levels (Reagent D), mitochondrial morphology and mitochondrial mass (Reagent C). Carry out the following steps: 1. Identify objects of interest in the images Identify (segment) nuclei in the Blue channel image (Reagent A) Depending on the image quality, a pre-processing step (such as shading removal, de-noising, etc.) may be required to prepare images for better nuclear segmentation. 36

37 Apply a segmentation method to separate nuclei from background and each other, so that the segmentation result corresponds as closely as possible with the objects observed in the original image, as illustrated in Figure 15 (adjust image visuals to clearly see nuclei in the original image). Figure 15. A Original image of Reagent A staining. B Result of nuclei segmentation (blue - nuclear mask) If required, use post-processing steps, such as erosion/ dilation, fill holes, sieve, and clump breaking, to improve segmentation results (improve position of outlines and separation between closely located nuclei; remove debris, etc.) Generate Cytoplasmic sampling region Using tools available in your image analysis software package, define a cytoplasmic sampling region. For example, based on the defined nuclear mask, a collar (a ring around nuclei, as demonstrated in Figure 16) can be generated around the nucleus to create a cytoplasmic sampling region. 37

38 Figure 16. A Nuclei image (Reagent A) - a collar sampling region (green outline) was generated on the basis of the nuclear mask (blue outline); B - Fused image of all 3 reagents; cytoplasm sampling region indicated by green outline Identify mitochondria using the Orange channel image (Reagent C) Apply a segmentation method to separate mitochondria (presented in the image as regions of granular or threadlike staining within cytoplasm) from background. Figure 17. A - Fused image showing nuclei (blue), intracellular calcium (green), and mitochondrial staining (red); B Orange channel image only (mitochondrial staining); C Mitochondria segmentation (mitochondria magenta mask; cytoplasmic sampling region yellow outlines). 2. Collect cell health information by applying the following measurement to cells: 2.1. Cell count, nuclear morphology, and intensity of staining Use nuclear mask to count all nuclei and all viable nuclei Use nuclear mask to measure morphological parameters from nuclei (e.g. nuclear area and form factor) and total intensity of nuclei staining as an indicator of cellular DNA content. 38

39 2.2. Calcium status Use cytoplasmic sampling region to measure intensity of calcium signal as an indicator of intracellular calcium levels Mitochondrial health Using mitochondria mask, collect measures from the Orange channel image (Reagent C), such as Total organelle area as an indicator of mitochondrial mass, organelle form factor as an indication of mitochondrial morphology, and so on Assay 2-2-Reagent viability assay Overview Based on fluorescent properties of the kit components and imaging capabilities of the majority of fluorescence imaging devices, images from this assay will be collected into 2 channels. Reagent Excitation/ Emission peaks (nm) Output image Objects identified Report A 355/465 Blue Channel All Nuclei Cell count Nuclear morphology and DNA content B 540/608 Orange Channel Dead cells nuclei % Viability Table 7. Description of assay reagents including spectral properties, output channels, organelles identified and output measures (2-Reagent assay set up). 39

40 Analysis Workflow Images are analyzed by defining nuclei based on the nuclear stain (Reagent A) detected in the Blue channel image. The resulting nuclear mask is applied to the Orange channel image (Reagent B) to identify dead cells. Live and dead cells can be counted and the percentage of viable cells reported. The following steps are recommended for the analysis : 1. Identification of objects of interest in the images Identify (segment) nuclei in the Blue channel image (Reagent A) Depending on the image quality, a pre-processing step (such as shading removal, de-noising, etc.) may be required to prepare images for better segmentation Apply a segmentation method to separate nuclei from background and each other, so that the segmentation result corresponds as closely as possible with the objects observed in the original image, as illustrated in Figure 18 (adjust image visuals to clearly see nuclei in the original image). Figure 18. A Original image of Reagent A staining. B Result of nuclear segmentation (blue- nuclei mask). 40

41 If required, use post-processing steps, such as erosion/ dilation, fill holes, sieve, and clump breaking to improve segmentation results (improve position of outlines and separation between closely located nuclei; remove debris, etc.). 2. Define live-cell and dead-cell sub-populations Using the nuclear mask generated in step 1.1, measure Average Intensity of signal from Reagent B (Orange channel), i.e. intensity of Reagent B signal will only be measured in the region of nuclei Reagent B selectively stains dead cells (cells with compromised plasma membrane only). Set up a threshold for signal B Average Intensity to separate live and dead cells, as shown in Figure

42 Figure 19. Identification of dead cells. A Nuclear staining (Blue Channel); B Image of dead-cell nuclei (Orange channel); C A threshold (vertical blue line) is set for nuclear average intensity in the Orange channel image to separate dead and live cells; D Data table (left) displays nuclei average intensity measured in the Orange channel image (reagent B); highlighted values correspond to nuclei as indicated by yellow arrows. Note that the value for the dead cell nucleus (red) falls above the set threshold ( vs ) Report percentage of live and dead cells as indication of cell viability Image Analysis - IN Cell Investigator For IN Cell users: a detailed description of analysis in IN Cell Investigator, an example Level 2 analysis protocol and a Level 1 context module are available for download from 42

43 10. Data handling Once images are analyzed, data can be further processed to generate IC50 curves and to perform high content analysis such as profile and cluster analysis as demonstrated below. Figure 20. Example data obtained with Cytiva Cell Health kit on HepG2 cells treated with amiodarone, nifedipine and sunitinib, over 72 hours. Images were acquired using IN Cell Analyzer 2000 using a 40 objective and analyzed using IN Cell Investigator, data post-processing was performed using Spotfire. A Cluster analysis of an example toxicity assay performed using the Cytiva Cell Health kit reveals dose-dependent effects of test substances on cellular phenotype. Profiles presented in the heat map are derived from 8 measured cellular parameters, relating to nuclear morphology, intracellular calcium levels, mitochondrial status and cell viability. B Parallel axis profile plots, based on the same parameters presented in panel B, aid in visualization of dosedependent compound effects. Profiles at 0 μm (green) and 12.5 μm (red) are compared for amiodarone (top), nifedipine (middle), and sunitinib (bottom). Two replicates are shown for each treatment condition. C Fused images of HepG2 cells treated with amiodarone, nifedipine and sunitinib, over 72 hour Cell nuclei (blue), cell mitochondria (red), Intracellular calcium (green) and dead-cell nuclei (red). 43

44 11. Troubleshooting guide Problem Overly confluent wells resulting in nuclear and mitochondrial segmentation problems during image analysis. Fluorescence intensity of images varies greatly across the cell assay plate. Mitochondrial parameters are unexpectedly high in treated wells where cytotoxicity is observed. Solution 1. Seed cells at a density that results in a monolayer of cells amenable to image analysis. 2. If available on your HCA platform, use optical sectioning during image capture. 1. Ensure dye reagents are mixed thoroughly and ensure multichannel pipette is delivering equal volume of dye across each channel. 2. Check that instrument is operating within normal parameters 1. Optimize nuclear segmentation parameters in the Blue channel. If nuclear segmentation is not optimal, some nuclei (particularly those of dead cells) may not be identified. When the nuclear mask is then applied to the Orange channel image to identify dead cell nuclei, some of Reagent B-positive signal is not seen as a signal derived from nuclei but mistakenly segmented as mitochondria leading to increased values in mitochondrial measures. 2. Adjust the live/dead filter threshold. Image analysis live/dead filter is not adequately removing dead cells from further analysis. The consequence of 44

45 Problem Mitochondrial parameters are unexpectedly high in treated wells where cytotoxicity is observed. (continued) Fluorescence intensity measured from Green Channel image lower than expected. Fluorescence intensity across plate appears inconsistent for more than one dye Fluorescence intensity not sufficient for adequate segmentation and analysis of resulting images. Solution this is that fluorescence deriving from Reagent B-positive nuclei can be miscategorized as being mitochondrial. 3. Optimize mitochondrial segmentation and avoid including dead cell nuclei using size and intensity filters. 1. Serum present in culture media can deactivate Reagent D. Once Reagent D is added to media it should be applied to cells as soon as possible. 2. Low signal could be reflective of the biology if none of the compounds tested exhibit significant cytotoxicity. A positive control such as Ionomycin can be included to confirm dye performance. 1. On addition to media ensure that a thorough mixing step is included to ensure homogeneity of dye concentrations across the whole cell assay plate. 2. Ensure read time for the plate is less than 90 minutes. 1. Ensure that image capture is optimized before the plate is scanned. Select several untreated and treated wells during the image optimisation step to ensure representative staining of dyes is adequately captured across the plate. 45

46 12. References 1. LIN, Z., et al. Toxicological Sciences, 126, pp , LUNDHOLT, BK., et al. J Biomol Screen, 8, pp REIS, Y., et al. PLoS ONE, 7, e ROQUEMORE, L., EuroBiotech, N11-12, Vol. 9, pp.34-40,

47 13. Related Products Product 47 Product Code Cytiva Cardiomyocytes Cytiva Cardiomyocytes 1e6 cells IN Cell Analyzer 6000 IN Cell Analyzer 6000 System (NA Only) IN Cell Analyzer 6000 System IN Cell Analyzer Environment Control Module IN Cell Analyzer Liquid Handling Module IN Cell Analyzer Temperature Control Module IN Cell Analyzer Transmitted Light Module IN Cell Analyzer Live Cell Package A (includes Temperature Control and Environment Control) IN Cell Analyzer Live Cell Package B (includes Temperature Control Liquid Handling) IN Cell Analyzer Live Cell Package C (includes Temperature Control, Liquid Handling and Environmental Control) IN Cell Analyzer 2000 IN Cell Analyzer 2000 Large Camera System (NA Only) IN Cell Analyzer 2000 Large Camera System IN Cell Analyzer 2000 Standard Camera System (NA Only) IN Cell Analyzer 2000 Standard Camera System IN Cell Analyzer Environment Control Module IN Cell Analyzer Liquid Handling Module IN Cell Analyzer Temperature Control Module IN Cell Analyzer Transmitted Light Module IN Cell Analyzer Live Cell Package A (includes Temperature Control and Environment Control) IN Cell Analyzer Live Cell Package B (includes Temperature Control Liquid Handling) (includes Temperature Control, Liquid Handling and Environmental Control)

48 GE Healthcare offices: GE Healthcare Bio-Sciences AB Björkgatan 30, Uppsala, Sweden GE Healthcare Europe GmbH Munzinger Strasse 5, D Freiburg, Germany GE Healthcare Bio-Sciences Corp. 800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ , USA GE Healthcare Japan Corporation Sanken Bldg , Hyakunincho, Shinjuku-ku, Tokyo , Japan For your local office contact information, visit GE Healthcare UK Limited Amersham Place Little Chalfont, Buckinghamshire, HP7 9NA, UK imagination at work AA 09/

49 Cell Health Assay 96-Well Plate Bring all reagents to Room Temperature (>18 C). Ensure your imaging system is in working order. Check sufficient storage space for images 4 C -20 C A B C D A B C D Spin tubes in microcentrifuge. Add dye reagents to media and mix well. Note: Add to cells within 15 minutes of preparing the 4 Dye Mix A 25 µl B 25 µl C 25 µl D 25 µl 6 ml Add 4 Dye mix to cells in culture plate. Caution: Correct addition of dye to plate is important for consistent assay performance. Incubate dye with cells for 1 hour at 37 C. Optimize Image capture and scan plate. Note: For optimal performance, the plate scan should be completed within 90 minutes following the 1 hour incubation step. < 90 mins Confirm images and associated files present in storage location. Optimize analysis parameters for object segmentation and live/dead filter. Run analysis and collect measurements relating to cell viability, nuclear morphology, mitochondrial status and calcium homeostasis.

50 Cell Health Assay 384-Well Plate Bring all reagents to Room Temperature (>18 C). Ensure your imaging system is in working order. Check sufficient storage space for images 4 C -20 C A B C D A B C D Spin tubes in microcentrifuge. Add dye reagents to media and mix well. Note: Add to cells within 15 minutes of preparing the 4 Dye Mix A B C D 50 µl 50 µl 50 µl 50 µl 12 ml Add 4 Dye mix to cells in culture plate. Caution: Correct addition of dye to plate is important for consistent assay performance. Incubate dye with cells for 1 hour at 37 C. Optimize Image capture and scan plate. Note: For optimal performance, the plate scan should be completed within 90 minutes following the 1 hour incubation step. < 90 mins Confirm images and associated files present in storage location. Optimize analysis parameters for object segmentation and live/dead filter. Run analysis and collect measurements relating to cell viability, nuclear morphology, mitochondrial status and calcium homeostasis. imagination at work GE, imagination at work and GE monogram are trademarks of General Electric Company. Cytiva is a trademark of GE Healthcare Companies General Electric Company-All rights reserved. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available upon request. Contact your GE Representative for the most current information. GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK AA 09/2012