A simple and robust cell-based assay for the discovery of novel cytokinesis inhibitors
2Department of Neuroscience, The Scripps Research Institute, Jupiter, FL 33458, USA
3WHM Consulting, LLC, 111 Sterling City Rd., Lyme, CT 06371, USA
Reagents, media and solutions
- Cultured cells (COS-7 are used here as example. However, any cell that undergoes cytokinesis in culture is expected to be amenable to this protocol. COS-7 Cat. # CRL-1651, American Type Culture Collection, Manassas, VA)
- Dulbecco’s Modified Eagle Medium (Cat. # 11995073, Life Technologies, Carlsbad, CA)
- Fetal Bovine Serum (Cat. # 26140079, Life Technologies, Carlsbad, CA)
- Antibiotic-Antimycotic solution (Cat. # 15240062, Life Technologies Carlsbad, CA)
- 75 cm2 flasks (Cat. # 430641U, Corning, Corning, NY)
- Dulbecco’s phosphate-buffered saline (PBS, Cat. # 14190250, Life Technologies, Carlsbad, CA)
- Trypsin-EDTA (0.25%) solution (Cat. # 25200072, Life Technologies, Carlsbad, CA)
- 96-well cell culture plates (Cat. # 25109, Genesee Scientific, El Cajon, CA)
- Jasplakinolide (Cat. # 2792/100U, R&D Systems (Minneapolis, MN)
- Cytochalasin D (Cat. # 113305, Cayman Chemical, Ann Arbor, MI)
- Swinholide A (Cat. # 501146229, Fisher Scientific, Hanover Park, IL)
- Blebbistatin (Cat. # 13013, Cayman Chemical, Ann Arbor, MI)
- para-aminoblebbistatin (Cat. # 22699, Cayman Chemical, Ann Arbor, MI)
- para-nitroblebbistatin (Cat. # 24171, Cayman Chemical, Ann Arbor, MI)
- Dimethyl sulfoxide (DMSO, Cat. # D2650, Sigma-Aldrich, St. Louis, MO)
- Solvent resistant polypropylene microplates (Cat. # 3357, Corning, Corning, NY)
- Fluorescein diacetate (FDA, Cat. # F7378, Sigma-Aldrich, St. Louis, MO)
- Hoechst33342 (Cat. # H3570, Life Technologies, Carlsbad, CA)
- Propidium iodide (PI, Cat. # P3566, Life Technologies, Carlsbad, CA)
- Culture medium: 89% Dulbecco’s Modified Eagle Medium, 10% Fetal Bovine Serum, and 1% Antibiotic-Antimycotic solution.
- Refrigerated centrifuge (Cat. # 5430 R, Eppendorf, Hauppauge, NY)
- Microplate shaker (Cat. # 12620-926, VWR, West Chester, PA)
- Fluorescence microscope (e.g., IN Cell Analyzer 6000 automated fluorescence microscope, Cat. # 29043323, GE Healthcare BioSciences, Marlborough, MA)
- Image analysis software (e.g., IN Cell Developer Toolbox software, Cat. # 25809826, GE Healthcare Bio-Sciences, Marlborough, MA)
1.Thaw frozen aliquots of cell line of interest and immediately dilute ten-fold in culture medium.
2.Centrifuge the cell suspension at 7197 × g at 20°C for 10 min in a refrigerated centrifuge.
3.Discard the supernatant and resuspend the pellet in culture medium at a final density of ~50000 cells/ml.
4.Plate cells onto 75 cm2 flasks at a density of 500000 cells/flask.
5.Following 3 d of incubation (37°C and 5% CO2), remove old media from the flasks and wash the cell layers twice with 5 ml of PBS.
6.Add 2 ml of Trypsin-EDTA (0.25%) solution to each flask to dissociate cells.
7.Incubate flasks at 37°C for 10 min to allow detachment of cells from the surface.
8.Cell dissociation can be further facilitated by pipetting the suspension up and down several times until no cell aggregates are observed by visual inspection under a stereomicroscope.
9.To inhibit trypsin, combine 8 ml of fresh culture medium with 2 ml of cell suspension.
10.Determine cell density by counting the cells in a hemocytometer.
11.Dilute the suspension to a density of 20000 cells/ml.
12.Immediately plate cells onto flat bottom, 96-well cell culture plates by transferring 100 µl of suspension to each well using a multichannel pipette, resulting in a final surface density of 2000 cells/well.
13.Following 24 h of incubation (37°C, 5% CO2), treat cells with compounds of interest prepared in compound plates.
14.Here, jasplakinolide, cytochalasin D, swinholide A, blebbistatin, para-aminoblebbistatin or para-nitroblebbistatin were first dissolved in DMSO.
15.Six-step serial 1:2 dilutions of compound solutions were then prepared in DMSO using solvent resistant polypropylene microplates. Concentration ranges (125–3.91 nM, 500–15.63 nM, 10–0.31 nM, 10–0.31 µM, 10–0.31 µM, and 10–0.31 µM for jasplakinolide, cytochalasin D, swinholide A, blebbistatin, para-aminoblebbistatin and para-nitroblebbistatin, respectively) were determined based on preliminary results. A 2 mM solution of para-aminoblebbistatin in DMSO was also prepared and used as positive control, while pure DMSO was used as a negative control.
16.Prepare compound plates by transferring 2.4 µl of positive and negative controls and compound solutions into each well of a 96-well plate containing 117.6 µl of culture media (50-fold dilution) using a multichannel pipette. Wells in the first and last rows should be used exclusively for negative and positive controls.
17.Mix solutions by shaking the compound plate for 1 min at room temperature at 1200 rpm using a microplate shaker. A typical plate layout map used for screening experiments is shown in Figure S1. Carry out all measurements in triplicate.
18.Transfer 100 µl of diluted compound solutions from the compound plate to the assay plate (containing cell cultures in 100 µl of culture media) using a multichannel pipette (2-fold dilution).
19.Following 24 h of incubation in the presence of compounds (37°C, 5% CO2), stain cells with FDA, Hoechst33342 and PI in a single step.
20.Prepare staining solution by combining 9 µl FDA (24 mM stock solution in DMSO), 22.2 µl Hoechst33342 (16.2 mM stock solution in water), 96 µl PI (1.5 mM stock solution in water) and 11.873 ml culture medium in a 50 ml centrifuge tube for each plate.
21.Use a multichannel pipette to add 100 µl of staining solution to each well of the assay plate containing 200 µl of treated cell culture, resulting in a final concentration of 6 μM, 10 μM, and 4 μM for FDA, Hoechst33342 and PI, respectively.
22.Incubate assay plates for 10 min (37°C, 5% CO2).
23.Replace the staining solution with fresh culture medium in each well (100 μl/well).
24.Lasers operating at 405 nm, 488 nm, and 561 nm in conjunction with 455 nm, 525 nm and 605 nm emission filters are used for the visualization of Hoechst33342, fluorescein and PI signals, respectively.
25.Identify and quantify nuclei of living and dead cells by using the corresponding fluorescent signal of Hoechst33342. Cell bodies of living cells are identified by using the corresponding fluorescent signal of fluorescein. Nuclei of dead cells are identified by using the corresponding fluorescent signal of PI.
26.If using the IN Cell Developer Toolbox software, nuclear segmentation (Hoechst33342) can be performed using the following parameters: minimum target area: 85 µm2, sensitivity: 50, sensitivity range 1.30, precise mask: enabled. Steps of postprocessing: watershed clump breaking, erosion (kernel size 9), sieve (keep targets with an area greater than 20 µm2), border object removal. Cytoplasm segmentation (fluorescein) can be performed using the following parameters: noise suppression: heavy, remove shading artifacts with area greater than: 2960 µm2, use octagonal morphology: enabled. Steps of postprocessing: sieve (keep targets with an area greater than 200 µm2), erosion (kernel size 3), watershed clump breaking, fill holes, dilation (kernel size 3), sieve (keep targets with an area greater than 200 µm2), border object removal. Nuclear segmentation (PI) can be performed using the following parameters: minimum target area 52 µm2, sensitivity: 20, sensitivity range 1.30, precise mask-enabled, use octagonal morphology: enabled. Steps of postprocessing: watershed clump breaking, erosion (kernel size 5), sieve (keep targets with an area greater than 20 µm2), border object removal.
27.A cytokinesis inhibitor allows nuclei to divide, but blocks the separation of cell bodies, resulting in the formation of bi- and multinucleated cells. The inhibitory effect of compounds can be estimated and compared by calculating the ratio of nuclei to cell numbers in the living cell population. Each living cell must contain at least one nucleus and each nucleus must belong to a living cell in this population. Therefore, fluoresceinpositive objects not overlapping with Hoechst33342-positive objects (potentially misidentified cells) and Hoechst33342-positive objects not overlapping with fluorescein-positive (dead and potentially misidentified nuclei) should be excluded from the calculation of the nuclei-to-cell ratio (NCR). This step was crucial to avoid several artifacts (see Anticipated results and troubleshooting and Discussion).
28.In addition to cytokinesis inhibition, compounds may show cytotoxic effects, which can affect the primary signal (e.g., through nuclear fragmentation or selective death of multinucleated cells). Therefore, cytotoxicity should also be quantified as the ratio of dead nuclei to total nuclei. Dead nuclei must show positive staining for both Hoechst33342 and PI. Therefore, all those objects not showing double labeling should be excluded from this calculation. Similarly, the total number of nuclei are calculated as the sum of Hoechst33342-positive objects overlapping with fluorescein-positive objects (living nuclei) and the Hoechst33342-positive objects overlapping with PI-positive objects (dead nuclei).
29.Plot the NCR against the compound concentration. To determine the half maximal effective concentration (EC50), fit the 6 point dose response data to the Hill equation:
Anticipated results and troubleshooting
|Eggert et al. ||Kepiro et al., Varkuti et al. [14,15]||Current protocol|
|Dyes used||Tetramethylrhodamine-NHS ester (total cytoplasm) Hoechst33342 (DNA/nuclei)||None||Fluorescein diacetate (living cells) Hoechst33342 (DNA/nuclei) Propidium Iodide (DNA/dead nuclei)|
|Fixing and staining time||~90 min, 6 steps||No||Staining only, ~15 min, 2 steps|
|Genetic labeling||None||EGFP-α-tubulin (cytoplasm) mCherry-H2B (nuclei)||None|
|Risk of cell loss||High||None||Low|
|Applicability to other cell lines||Yes||Genetic labeling is needed first||Yes|
|Microscopy||Automated fluorescence microscopy||Manual||Automated fluorescence microscopy|
|Image processing||Automated counting of nuclei Manual counting of binucleated cells||Manual||Fully automated (Highly resistant to artifacts)|
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