A high-content assay for neurite outgrowth using the IN

Remove antibody, wash the cells with 200 µl of PBS. 14. Dilute the Goat anti-mouse IgG Cy3, Cy5, Texas Red, and FITC-linked antibodies 1:5000 in PBS, ...

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IN Cell Analyzer 2000

A high-content assay for neurite outgrowth using the IN Cell Analyzer 2000 Key words: Neurite outgrowth • fluorescent image analysis • IN Cell Analyzer 2000 • Preview scan • IN Cell Investigator • high-content analysis • cell imaging Neurite outgrowth plays a fundamental role in embryonic development, neuronal differentiation, and nervous system function. The process is also critical in some neuropathological disorders as well as neuronal injury and regeneration. The extracellular environment, as well as biological and pharmacological agents, can affect a variety of neurite features, such as the length and number of neurites per cell. High-content neurite outgrowth assays allow direct screening of the morphological effects of various treatments, and can be multiplexed with additional structural and biochemical probes to increase information content. In this assay, neuro-2a (N2a) cells are treated with increasing concentrations of retinoic acid to stimulate outgrowth of neurites. The protocol requires use of at least two fluorescent labels: one to mark cell nuclei and a second to identify the entire cytoplasm of the cell (including neurites). To allow maximum flexibility for multiplexing, the assay has been validated for use with secondary antibody conjugated to FITC, Cy3™, Texas Red™ or Cy5™. Images were acquired with the IN Cell Analyzer 2000 and analyzed using IN Cell Investigator software to quantitate mean neurite length per cell. During the course of the study, it was observed that at higher drug concentrations, many target cells became detached, and of those that remained, a large proportion tended to cluster around the well edges. In multiwell assays, artifacts such as this could lead to aberrant results if the imaging position cannot be optimized prior to image capture. For example, using a central position for image capture could lead to under-reporting of structural features and unacceptably high variance between replicate wells. The Preview scan function introduced with the IN Cell Analyzer 2000 system helps avoid such artifacts by

allowing the user to quickly preview the entire well content, and then choose the optimal imaging position of multiple fields of view prior to imaging the entire plate.

Materials Products used IN Cell Analyzer 2000, standard chip CCD camera 28-9534-63 IN Cell Investigator Software, single seat license 28-4089-71 Goat anti-mouse IgG, Cy3-linked, 1 mg PA43002 Goat anti-mouse IgG, Cy5-linked, 1 mg PA45002

Other materials used Neuroblastoma neuro-2a cells ATCC, CCL-131 Eagle’s Minimum Essential Medium (EMEM) ATCC, 30-2003 Penicillin-streptomycin (P-S) 100× Sigma, P4333 (penicillin: 10 000–12 000 units/ml; streptomycin: 10–12 mg/ml) L-glutamine 200 mM solution Sigma, G7513 Fetal bovine serum (FBS) gold PAA, A15-151 Triton™ X-100* Sigma, T8787 PBS Sigma, D8537 Formalin solution, neutral buffered, 10%* Sigma, HT5012 Monoclonal anti-α-tubulin antibody, clone DM1A Sigma, T9026 Goat anti-mouse IgG-FITC-linked Sigma, F2012 Goat anti-mouse IgG-Texas Red-linked Invitrogen, T-6390 Retinoic acid* Sigma, R2625 Hoechst™ 33342 (5 µM final) Invitrogen, H21492 Ethanol 100% Hayman chemicals DMSO Sigma, D2650 Sterile water Fresenius Kabi, 22-96-985 µClear™ plates, 96-well tissue culture treated, black Greiner Bio One, 655090 GraphPad Prism™ 4.0 GraphPad Software * Triton X-100, formalin solution, and retinoic acid are classified as harmful. Handle in accordance with MSDS and local laboratory safety guidelines.

Cell growth medium

Imaging on IN Cell Analyzer 2000

Minimum Essential Medium Eagle’s media supplemented with fetal calf serum 10%, L-glutamine and penicillin-streptomycin.

For maximal detection of neurites, it is advisable to choose a low power objective (e.g., 10×), Since neurite morphology can vary dramatically with drug treatment, the Software Autofocus function was chosen to optimize the focal plane for neuriteextended and non-neurite extended cells.

Reduced-serum medium Minimum Essential Medium Eagle’s media supplemented with fetal calf serum 2%, L-glutamine and penicillin-streptomycin.

Methods To induce neurite outgrowth, serum-starved N2a cells were incubated with retinoic acid at a range of dilutions. Cells were then fixed, permeabilized, and stained with anti-α-tubulin primary antibody followed by addition of secondary antibody conjugated to a fluorescent dye. Cell nuclei were stained with Hoechst 33342. Cells were imaged using IN Cell Analyzer 2000 and quantitated using a user-defined protocol created with the IN Cell Investigator image analysis software.

Assay protocol 1. Maintain cells in cell growth medium, passaging them at 75% to 80% confluence. 2. Seed N2a cells in reduced-serum medium into 96-well µClear plates at ~ 12 000 cells/well (100 µl) and incubate under standard tissue culture conditions for 5 h. 3. Using an initial 10 mM retinoic acid concentration, prepare a dilution series of 200 µM (50× dilutions) to 20 µM in reduced-serum medium. 4. Add 50 µl of reduced-serum medium to each well. 5. Add 50 µl of retinoic acid at various concentrations to appropriate wells (quadruplicate samples). Final retinoic acid concentration range is 50 µM to 5 µM. Add 50 µl of buffer to the control wells. 6. Incubate under standard tissue culture conditions for 18 h or overnight. 7. Add 150 µl of 10% formalin (~ 4% formaldehyde) and allow the cells to fix at room temperature for 2 h. 8. Remove formalin from all wells and wash the cells with 200 µl of PBS. Repeat the wash step. 9. Add 200 µl of PBS per well. Plates can be stored at 4ºC at this stage. 10. Remove PBS, add 100 µl 0.1% Triton X-100 per well. 11. Remove Triton X-100, wash the cells with 200 µl of PBS. 12. Dilute the monoclonal anti-α-tubulin antibody 1:1000 in PBS, add 100 µl per well. Incubate at 37ºC for 1 h. 13. Remove antibody, wash the cells with 200 µl of PBS. 14. Dilute the Goat anti-mouse IgG Cy3, Cy5, Texas Red, and FITC-linked antibodies 1:5000 in PBS, add 100 µl to the appropriate wells. Incubate at 37ºC for 1 h. 15. Add 150 µl of PBS, and then add 50 µl of 20 µM Hoechst 33342 per well (5 µM final). 16. Store the plates protected from light at 4ºC.

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1. Turn on the lamp. 2. Switch the automated polychroic changer to the QUAD2 position for the Texas Red defined protocol, or to the QUAD1 position for the Cy3 defined protocol. 3. Enter the Assay Development Mode and create a new Protocol Designer, Work through the Wizard to generate an imaging protocol. 4. Enter the description for your imaging experiment. 5. Select an objective from the dropdown menu; for this application the Nikon™ 10×/0.45 NA objective was selected. 6. Select µClear from the Plate type dropdown menu. 7. Within Microscopy, select the number of wavelengths to be used and the binning option. 8. Select the excitation and emission filter sets for the fluorophores under investigation; to detect Hoechst nuclear stain, select filter set DAPI_DAPI (350/50× and 455/50m), for FITC select FITC_FITC (490/20× and 525/36m), for Cy3 select Cy3_Cy3 (543/22× and 605/64m), for Texas Red detection select TexasRed_TexasRed (579/34x and 624/40m), and for Cy5 select Cy5_Cy5 (645/30×_705/72m). 9. Select the image processing option and exposure time. 10. Enter Plate/Slide View and select the wells for acquisition. 11. Click within a selected well to activate the imager. 12. Return to the Wizard and within Focus select the Software Autofocus strategy. 13. Optimize the exposure setting and focus position for each wavelength. 14. Optimize the imaging position as shown in Fig 1a. Activate the Preview scan and select the region of interest (b) Run Preview scan, (c) Select the required number of fields, and (d) Position the selected fields over the cell area. 15. Enter Acquisition Mode and Image the sample plate(s).

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Fig 1. Utility of the Preview scan function within IN Cell Analyzer 2000 allows the user to optimize the imaging position of multiple fields of view: (a) Selection of region of interest for Preview scan; (b) Preview image showing Hoechst 33342-stained nuclei over the entire area of the well; (c) Adjustment of fields from the center of the well to the periphery; (d) Location of the imaging position over an area of uniform cell growth.

4. Create a target set called Neurites, use a preprocess macro to subtract cell bodies from whole cells to generate a neurites mask (Fig 2g), apply to the channel containing the images of antibody-stained neurites (Fig 2f). 5. Create a One-to-Many targets set (cells with neurites) to link Cell body (primary target) with Neurites (secondary target set). Set overlap conditions as required (e.g., any intersection – secondary target within primary target). To count each neurite only once, uncheck the option Allow multiple primary targets to share secondary targets. 6. This protocol can be used to make population and cell-bycell measurements such as neurite length/cell; neurite count/ cell and total neurite length/cell. Here, we have measured mean neurite length per cell.

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Analysis protocol The strategy for analysis is to identify neurites by subtracting a binary image of cell bodies (derived by eroding a whole-cell binary image to exclude neurites) from a binary image of the entire cell (including neurites). In conjunction, information from the nuclear channel is used to derive individual cell data. As outlined below, IN Cell Developer Toolbox can be used to create an analysis routine based on this strategy. Generation of this algorithm is described elsewhere (1). In brief: 1. Open the image stack in the IN Cell Developer Toolbox. 2. Create a target set called Nuclei and segment the objects within the Hoechst channel to identify nuclei (Fig 2a). Measure the total count of nuclei in the population. 3. Create a target set called Cell body, segment the objects to outline the whole cell including neurites (Fig 2b), and direct the binary image to a separate channel (Fig 2c). Erode the segmented objects to create a cell bodies mask (Fig 2d), direct the binary image to a separate channel (Fig 2e). Dilate the final cell body image to enable overlap with neurites.

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Fig 2. Neurite outgrowth analysis workflow in the IN Cell Developer Toolbox; (a) Segmented nuclei mask, (b) Segmented whole cells mask and (c) Corresponding binary image, (d) Segmented cell bodies mask and (e) Corresponding binary image before final dilation step, (f) Segmented neurites mask, (g) Binary images of neurites generated from (c) minus (e).

Results Using IN Cell Analyzer 2000, images were acquired from four neurite outgrowth assay plates (each using a different dyeconjugated secondary antibody: FITC, Cy3, Texas Red, or Cy5). Image quality was of a high standard in terms of feature resolution (Fig 3). Background signal in the images was also low, which contributed to good gray-level contrast (Fig 4a). (a)

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Fig 3. Fused color image acquired with IN Cell Analyser 2000 from mouse retinoblastoma N2a cells treated with retinoic acid to induce neurite outgrowth. Cell bodies and neurites are labeled with Goat anti-mouse IgG conjugated to Cy5 (red). Nuclei are stained with Hoechst 33342 (blue).

Analysis of the image stacks was performed using the IN Cell Developer Toolbox user-defined protocol as described, to identify nuclei and neurites (Fig 4b), reporting mean neurite length per cell.

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Fig 4. (a) IN Cell Analyzer 2000 gray-scale image showing mouse N2a cells with retinoic acid-induced neurite extensions, (b) Segmentation outlines superimposed on the corresponding fused color image following analysis with IN Cell Developer: nuclei (blue outlines); dilated cell bodies (red outlines); neurites (green outlines).

From each analysis run, the data files were opened in GraphPad Prism software, plotting mean neurite length per cell vs retinoic acid concentration (Figs 5a to d). In each case, mean neurite length increased with an increased concentration of retinoic acid, with a downward deflection noted at high (> 40 µM) levels of drug. Each fluorophore gives a good dynamic range and an acceptable signal window (SSMD) of > 4.0 [2]. The results demonstrate that maximum neurite length is achieved with a retinoic acid concentration of 30 to 40 µM.

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(d) Fig 5. Mean neurite length per cell plotted as a function of retinoic acid concentration. Retinoic acid-treated N2a cells were stained with fluorophore conjugated to Goat anti-mouse IgG secondary antibody directed against anti-α-tubulin primary antibody. IN Cell Analyzer 2000 images were acquired with the 10× objective. Data points represent the mean +/- SEM of four replicate wells, nine fields of view per well. (A) Data generated from FITC labeled Goat anti-mouse IgG; (B) Data generated from Cy3 labeled Goat anti-mouse IgG, (C) Data generated from Texas Red labeled Goat anti-mouse IgG, (D) Data generated from Cy5 labeled Goat anti-mouse IgG. For each data set, SSMD (2) was calculated using the peak response data point and the 0 μM retinoic acid data point.



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For a direct comparison of data from all four labeled antibody studies, normalized mean neurite length/cell was plotted against the log retinoic acid concentration (Fig 6). This comparison demonstrated that each experiment gave similar response dynamics, with the graphs displaying similar curve shape and X-Y axis inflection position.

Fig 6. Mean neurite length per cell plotted as a function of retinoic acid concentration using Goat anti-mouse IgG-fluorophore staining of monoclonal anti-α-tubulin antibody treated N2a cells, (A) Data generated from FITC labeled Goat anti-mouse IgG, (B) Data generated from Cy3 labeled Goat anti-mouse IgG, (C) Data generated from Texas Red labeled Goat anti-mouse IgG, (D) Data generated from Cy5 labeled Goat anti-mouse IgG.

Since retinoic acid is known to be cytotoxic at high concentrations (3), for each data point, average neurite length per cell for four replicate wells was derived from a total of nine fields of view per well. In addition, the Preview scan function was used to optimize imaging position prior to acquisition. The resulting consistency of pharmacodynamic response across the four separate experiments (Fig 6) indicates that the assay is robust and reliable despite the cell number declining in a dose-dependent manner (Fig 7).

Fig 7. Measured decrease in cell number at higher concentrations of retinoic acid addition.

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Conclusions The study of neurite outgrowth is relevant to both drug discovery and elucidation of the mechanisms underlying neurite initiation and extension. Here, we have demonstrated the flexibility of IN Cell Analyzer 2000 in developing robust automated approaches for detection and quantitation of neurite outgrowth. The protocols presented are suitable for functional studies as well as screening applications. Preview scan allows the user to select the optimal imaging position prior to acquiring data from the entire plate, thus avoiding any under-reporting of structural features due to patchy cell distribution. Multiple filter sets and automated polychroic changing allow the imaging of neurite outgrowth using a wide range of fluorescent dyes and antibody conjugates, thereby adding flexibility when setting up multiplexing experiments with additional probes. The resultant image stacks are fully compatible with IN Cell Investigator image analysis software, generating relevant biological data at each of the wavelengths studied.

References 1.

Application note: Neurite outgrowth cell-by-cell analysis using the IN Cell Developer Toolbox, GE Healthcare, 14-0005-35, Edition AA (2005).

2.

Zhang, D. H. A pair of new statistical parameters for quality control in RNA interference high-throughput screening assays. Genomics 89(4), 552–561 (2007).

3.

Voigt, A. and Zintl, F. Effects of retinoic acid on proliferation, apoptosis, cytotoxicity, migration, and invasion of neuroblastoma cells. Medical and Pediatric Oncology 4, 205–213 (2003).

GE, imagination at work, and GE monogram are trademarks of General Electric Company. Cy3 and Cy5 are trademarks of GE Healthcare companies. The IN Cell Analyzer and associated analysis modules are sold under use licenses from Cellomics Inc. under US patent numbers US 5989835, 6416959, 6573039, 6620591, 6671624, 6716588, 6727071, 6759206, 6875578, 6902883, 6917884, 6970789, 6986993, 7060445, 7085765, 7117098; Canadian patent numbers CA 2282658, 2328194, 2362117, 2381334; Australian patent number AU 730100; European patent numbers EP 0983498, 1095277, 1155304, 1203214, 1348124, 1368689; Japanese patent numbers JP 3466568, 3576491, 3683591 and equivalent patents and patent applications in other countries. CyDye: This product or portions thereof is manufactured under an exclusive license from Carnegie Mellon University under US patent number 5,268,486 and equivalent patents in the US and other countries. The purchase of CyDye products includes a limited license to use the CyDye products for internal research and development but not for any commercial purposes. A license to use the CyDye products for commercial purposes is subject to a separate license agreement with GE Healthcare. Commercial use shall include: 1. Sale, lease, license or other transfer of the material or any material derived or produced from it.

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