Use of Drosophila S2 Cells for Live Imaging of Cell Division

Summary

Cell divisions can be visualized in real time using fluorescently tagged proteins and time-lapse microscopy. Using the protocol presented here, users can analyze cell division timing dynamics, mitotic spindle assembly, and chromosome congression and segregation. Defects in these events following RNA interference (RNAi)-mediated gene knockdown can be assessed and quantified.

Abstract

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:α-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as ‘CID’ gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.

Introduction

Cell Division is a process critical to all multicellular organisms, both in their development and homeostasis¹. Drosophila has long been used as a model for the study of cell division, with experiments in various tissue types and genetic conditions providing key insights into the process. While many of these insights come from fixed cell conditions, cell division is a dynamic procedure with many moving parts, making visualization of live cells integral to assessing RNAi or genetic knockout effects on numerous parts of cell division, including spindle formation, chromosome congressional and segregation, and cytokinesis.

Many protocols have been developed and utilized over the years to visualize Drosophila­ cell divisions in vivo. Various groups have cultivated techniques to image divisions in both larval imaginal discs and larval brains^{2,3,4,5,6,7,8}. These techniques, while useful for imaging in specific tissues, are limited in throughput and often require generation and maintenance of genetic stocks to generate fluorescent components and alter the expression of genes of interest. Cultured S2 cells from Drosophila provide a higher throughput alternative to rapidly test the effects of various genes in cell division. Furthermore, with the ability to transfect various fluorescent proteins, S2 cells can be quickly modified to determine the effects of RNAi knockdown on numerous components of cell division. Fluorescently tagged genes of interest can also be observed in cell division, allowing for dynamic characterization of their function⁹.

Here we provide a detailed protocol for live imaging of mitotic S2 cells using methods we recently described¹⁰. Our method utilizes stably transfected cells with fluorescent markers for microtubules and centromeres whose expression are under control of a copper-inducible promotor (metallothionein; pMT). This methodology can be used to image spindle and chromosome dynamics in all phases of mitosis utilizing a relatively simple fluorescent microscope with basic imaging software. It can be further customized to fit individual research needs, with transient transfection and RNAi offering expanded possibilities for determining the role of candidate genes in mitosis. Because of the relative simplicity of the protocol, it can be used for small-scale loss-of-function screens to identify genes for which further study in vivo would be beneficial, allowing for more focused efforts and efficiency with subsequent genetic manipulation in flies.

Protocol

1. Preparation of S2 Cells for Treatment with RNAi

1. From a stock culture of confluent pMT:GFP:CID and pMT:mCherry:α-tubulin stably transfected S2 cells (~80% viable; grown at 24-28 ˚C), seed cells in a 6 well sterile culture plate at a density of 1 x 10⁶ cells/mL in fresh, warmed, 10% fetal bovine serum (FBS) supplemented Schneider’s insect media (SIM).
   NOTE: Stably transfected S2 cells can be generated by following a protocol from the Drosophila RNAi Screening Center (DRSC) (https://dgrc.bio.indiana.edu/Protocols?tab=cells). Although the specific stable S2 line used herein utilizes GFP and mCherry, numerous alternatives exist both within these fluorescent spectra as well as others. A detailed discussion of these fluorescent proteins is beyond the scope of this protocol, but their properties and potential advantages/disadvantages have been expertly reviewed elsewhere ¹¹.
   1. Using a cell counter or hemocytometer, determine the density of confluent cell stock.
   2. Determine the volume of confluent cell suspension necessary to achieve 1 x 10⁶ cells/mL in a total volume of 4 mL (e.g., ~4 x 10⁶ total cells). Add this volume of cell stock to a volume of fresh SIM to make 4 mL/well total volume. Determine the density of the cell stock (cells/mL) by diluting 100 μL of cells directly from the stock flask with 100 μL of media and counting this 1:2 dilution either manually with a hemocytometer or with an automatic cell counter if available.
      NOTE: If using S2 cells not stably transfected, a transient transfection assay with fluorescently tagged (GFP, mCherry, etc.) α- or β-tubulin, and a DNA marker (CENP-A, Histone H2B, etc.) can be performed. Further, stably transfected cell lines with various mitotic spindle and DNA markers are available from the Drosophila Genetics Resource Center that may better suit the exact experimental needs of the user (https://dgrc.bio.indiana.edu/cells/Catalog).
   3. Place freshly seeded cells into 24-28 ˚C incubator for 36-48 h.

2. Treatment of Seeded Cells with dsRNA Against Gene(s) of Interest

NOTE: The following example and Results section will use Shortstop (Shot), an actin-microtubule crosslinking agent as the gene of interest. Use a mock treatment with no dsRNA or with dsRNA directed against an irrelevant gene (e.g., beta-galactosidase, lacZ) as a negative control.

1. Warm Schneider’s insect media not supplemented with FBS (Serum free media; SFM) in 24-28 ˚C incubator.
2. Transfer cells from the previously seeded well (from step 1.1.2) into a 15 mL sterile tube, noting the total volume of cells transferred.
   1. Retain a small volume of cells (~100 µL) for counting in a cell counter or hemocytometer.
3. Gently pellet cells by centrifuging at 1,000 x g for 3 min at room temperature.
   1. While centrifuging cells, determine the concentration of cells per mL using a cell counter or hemocytometer. Multiply this number by the total volume being centrifuged (noted in 1.2.2.) to obtain the total number of cells.
4. Aspirate the supernatant from the pelleted cells.
5. Re-suspend the cells in warmed SFM to obtain a concentration of 3 x 10⁶ cells/mL.
   1. Transfer 1 mL of the resuspended cells to a new well in a 6 well plate.
6. Add 10-50 µg of dsRNA (diluted in 100 μL of RNAase-free water) against Shot (or the target gene of interest) directly to the cells and swirl the plate to mix. Incubate the plate for 1 h in the 24-28 ˚C incubator to allow for direct dsRNA uptake into cells.
7. During the 1 h incubation, warm the 10% FBS supplemented SIM in the 24-28 ˚C incubator.
8. After incubating cells with dsRNA for 1 h, add 2 mL of 10 % FBS supplemented SIM directly to the well without removing the 1 mL of media.
9. Place cells into 24-28 ˚C incubator for 3-7 days.
   NOTE: As with dsRNA concentration, total treatment time may need to be optimized for the desired target gene. To evaluate RNAi efficacy, perform a western blot on the whole cell lysates using an antibody against the target gene. If the initial dsRNA target sequence proves ineffective, designing alternative dsRNAs targeted to unique sequences within the target gene.

3. Induction of Fluorescent Protein Expression and Preparation of Cells for Imaging

1. If stably transfected cells were generated using the pMT plasmid (containing an inducible metallothionein promotor) as described here, induce expression of fluorescent proteins by treating cells with copper sulfate (CuSO₄) at a final concentration of 500 µM for 24-36 h prior to imaging and 4 days after RNAi treatment. Use of constitutive expression plasmids such as pAct does not require copper induction.
2. Prepare Cells for Imaging
   1. Warm the 10% FBS supplemented SIM in the 24-28 ˚C incubator for 1 h.
   2. Transfer cells into a 15 mL sterile tube, noting the total volume of cells transferred.
      1. Retain a small volume of cells (~100 µL) for counting in a cell counter or hemocytometer.
   3. Gently pellet cells by centrifuging at 1,000 x g for 3 min at room temperature.
      1. While centrifuging cells, determine the concentration (cells/mL) using a cell counter or hemocytometer. Multiply this number by the total volume being centrifuged (noted in 2.2.2.) to obtain the total number of cells.
   4. Aspirate the supernatant from the pelleted cells.
   5. Resuspend the cells in fresh, warmed 10% FBS supplemented SIM to obtain a concentration of 2 x 10⁶ cells/mL. Add an appropriate amount of CuSO₄ to maintain 500 µM concentration.
   6. Transfer 200-500 µL of re-suspended cells to one well of a multi-well live cell chamber and place on the inverted fluorescent microscope. Allow the cells to settle in the chamber for 15-30 min prior to imaging experiments.
      NOTE: Live cell chamber wells can be pre-coated with poly-L-lysine for additional adherence.

4. Set Up a Live Cell Imaging Program

NOTE: Live Cell Imaging in this experiment was performed using an inverted imaging system and its associated software (e.g., Olympus IX83 with the cellSens Dimensions software package). Details will differ by microscope manufacturer and software package; thus, general guidelines and operations are listed below.

1. Using the software that runs the inverted fluorescent microscope, prepare a program for imaging a cell (or cells) over time.
   1. Open the software by double-clicking on the software desktop icon.
   2. Create a new experimental file by clicking File, then New experimental file.
   3. First, insert a time lapse loop over which images will be taken. Do this by clicking the Stopwatch icon (Time-lapse Loop icon) from the icon bar. Set the interval in s in the Experiment Manager tab, and then set the number of cycles in the same tab by dividing the desired overall experiment length (in s) by the interval. Allow the loop to repeat over the desired total time period.
      NOTE: Typically, images are taken every 30-60 s over a period of 3-4 h.
   4. Within the time lapse loop layer, insert an infrared focus check to maintain the focus of the objective (if available). To do this, add a Z-drift Compensation (ZDC) step within the Time-lapse Loop layer by clicking on the Square with Two Arrows icon (Move XY icon) and selecting Z-Drift Compensation from the dropdown menu.
      NOTE: The term infrared focus check refers to a system by which an infrared pulse is used to maintain a constant distance between the objective and the slide/imaging chamber. Many microscopes have such a system, each with their own proprietary nomenclature. Readers should consult their operation manual or representative for specific naming details.
   5. After the infrared focus check, add a step in the program to take a multichannel image (e.g., FITC for GFP and TRITC for mCherry) over a 3-5 z-stacks by first inserting a z stack step, then specifying the channel. Do this by adding a Multichannel Group layer within the Time-lapse loop layer by clicking on the Color Wheel icon (Multichannel Group icon). Next, add a Z-Stack Loop layer within the Multichannel Group layer by clicking on the 3-layered icon (Z-stack loop icon). Set the desired Step Size and Number of Slices in the Experiment Manager tab and set the exposure of each channel as low as possible to minimize photobleaching.
      NOTE: The z-stack interval, exposure time, and percent transmittance for LEDs can vary. Typical in these experiments, use three z-stacks taken over a range of 3 µm, giving an interval of 1 µm between each stack. Defining the center of the cell rather than the top and bottom tends to lead to the best results. Images are then collected for each channel(s) at an exposure of 50 ms and percent transmittance of 50% with no neutral density (ND) filter engaged.

5. Image Dividing Cells Using Live Cell Imaging Program

NOTE: S2 cells do not require CO₂ and grow optimally at 23-27 °C. All imaging was performed at ambient temperature in a well-controlled room.

1. Using the oculars, find the top (or bottom) corner of the well and focus the objective (40-60x oil immersion) onto the cells using the mCherry (α-tubulin) channel.
2. Scan along the top (or bottom) of the well to move away from the vertical well divider.
   NOTE: Imaging too close to the well dividers can interfere with infrared focus checks.
3. Find a cell (or cells) that are in late G2 or early M-phase (prophase).
   NOTE: These cells can be best identified by the existence of exactly two ‘star-like’ microtubule structures (centrosomes) and an intact nucleus (indicated by a circular region of refracted light within the cell), both easily distinguished using the α-tubulin (red) excitation filter. Selection of cells in earlier interphase, notable by one or zero easily visible centrosomes, can result in wasted time due to a lag in G2/M progression. Conversely, selection of cells after NEBD will prevent accurate calculation of mitotic timing and imaging of early spindle assembly and chromosome dynamics. Also, S2 cells often contain >2 centrosomes. Although these typically clusters into a bipolar spindle¹², it is advised that users avoiding these cells unless otherwise desired for the experimental design. Images and discussion of appropriate cells to select can be found in the Representative Results section.
4. Click the Live view button to begin viewing cells in the software screen. Using the fine focus knob of the microscope, focus the cell (or cells) of interest. Set the infrared focus check by clicking the Set Focus button.
5. Initiate the time lapse imaging program by clicking the Start button. Adjust the histograms as needed by selecting the desired channel and adjusting the mean pixel intensities to see the cell (or cells) of interest clearly.
6. Allow the program to run, checking the cell after 15-20 min to ensure nuclear envelope breakdown (NEBD) has occurred, which is determined by the disappearance of the round dark, a refracted spot near the cell center.
7. Continue allowing the program to run, checking intermittently (every 15-20 min) to determine once anaphase onset has occurred. Stop the program at this point if there is more time remaining in the total time period and save the file. Alternatively, if events during telophase and cytokinesis are of interest, allow the program to run a sufficient time in order to image these processes as well.
8. Perform analysis of NEBD to anaphase onset timing by noting the time of NEBD (t­_NEBD) in min and the time of initial chromosome separation (t_{anaphase onset}) in min and subtracting t­_NEBD from t_{anaphase onset} to obtain the NEBD to anaphase onset time for a given cell.
   1. Do this by clicking the frame up button and noting the frames where NEBD and anaphase onset occur, subtracting these to determine the total number of elapse frames, and multiplying this by the time interval between imaging frames.
9. Continue scanning for pre-dividing cells to obtain multiple n’s for a given condition. Cells can be imaged in the live cell chamber well for up to 12 h after their initial settling.

Representative Results

Methods we describe above will result in the identification and imaging of Drosophila S2 cells undergoing cell division. Cells that are about to divide (i.e., just prior to entering M-phase) can be targeted by the presence of two centrosomes and an intact nucleus indicated by refracted light and a darker spot within the cell when viewed in the α-tubulin channel (Figure 1, left-most panels, red channel; arrowhead in Figure 1A). We estimate that approximately 2-5% of cells fall into this category, and between imaging experiments, we typically spend a maximum of 2-3 minutes scanning for another qualified cell. NEBD can be visualized through the disappearance of this dark spot, resulting in the uniform coloring of the cytoplasm (Figure 1, panels marked “00:00”, red). After NEBD, the time each cell takes to form the spindle, congress chromosomes, and segregate chromosomes can be measured simply by noting the time points of these events relative to NEBD.

We utilize these divisions to assess mitotic timing, specifically of NEBD to anaphase onset, noting the point at which chromosomes begin to separate. A majority (~90%) of copper-induced cells, expressed both mCherry:α-tubulin and GFP:CID. A small percentage of cells (~10%) expressed only one marker or neither. These cells were avoided during cell selection. Typically, S2 cells display NEBD to anaphase onset timing of between 20-30 minutes (Figure 1A, Control). We next treated cells with dsRNA targeted against Shortstop (Shot), an actin-microtubule crosslinking protein we suspected may affect cell cycle dynamics. Indeed, following Shot knockdown cells exhibited a significant mitotic delay (Figure 1B, ShotRNAi delay), with many cells arresting at metaphase and never transitioning to anaphase during the entire 2-3-hour imaging experiment (Figure 1D, ShotRNAi arrest). We reasoned this delay/arrest phenotype could likely be due to activation of the spindle assembly checkpoint (i.e. the M-phase checkpoint). To directly test this hypothesis, we co-treated cells with dsRNA against Shot and Rough deal (Rod), an important component of this checkpoint¹³. This led to a suppression of the arrest phenotype, resulting in NEBD to anaphase times similar to Control (Figure 1C, ShotRNAi+RodRNAi). Thus, this live imaging protocol allowed us to conclude that Shot is required for timely M-phase progression and that its loss leads to checkpoint activation that delays or arrests mitotic cells.

Figure 1: Live imaging reveals cell cycle defects due to mitotic checkpoint activation in S2 cells.
In all conditions, live-cell imaging was conducted on S2 cells stably co-transfected with inducible GFP:CID and mCherry:αTubulin as described herein. (A) Cartoons illustrate important landmarks during mitosis, and corresponding images are shown from representative movies of indicated genotypes with time points relative to NEBD (t=00:00) indicated. Control cells typically progress to anaphase within 20-30 min. ShotRNAi-treated cells display NEBD-anaphase delay (B) and frequently undergo metaphase arrest, never entering anaphase within the imaging experiment (D). Co-treatment of cells with ShotRNAi and RodRNAi, a component of the spindle assembly checkpoint, suppresses the Shot phenotype leading to anaphase onset kinetics similar to Control cells (C). The arrows in (A) indicate the ‘star-like’ centrosome structures, and the large arrowhead indicates the nucleus. Each scale bar represents 5 microns. This figure is adapted and republished with permission from¹⁰ (https://www.molbiolcell.org/info-for-authors). Please click here to view a larger version of this figure.

Video 1: Time-lapse movie of ‘Control’ S2 cell division. Video shows a representative movie of S2 cell division in ‘Control’ genotype. This movie corresponds to Figure 1A. This video is adapted and republished with permission from¹⁰ (https://www.molbiolcell.org/info-for-authors). Please click here to download this video.

Video 2: Time-lapse movie of delayed ‘ShotRNAi’ S2 cell division. Video shows a representative movie of S2 cell division in ‘ShotRNAi’ genotype that leads to a phenotypic mitotic delay. This movie corresponds to Figure 1B. Please click here to download this video.

Video 3: Time-lapse movie of ‘ShotRNAi+RodRNAi’ S2 cell division. Video shows a representative movie of S2 cell division in ‘ShotRNAi+RodRNAi’ genotype. This movie corresponds to Figure 1C. This video is adapted and republished with permission from¹⁰ (https://www.molbiolcell.org/info-for-authors). Please click here to download this video.

Video 4: Time-lapse movie of arrested ‘ShotRNAi’ S2 cell division. Video shows a representative movie of S2 cell division in ‘ShotRNAi’ genotype that leads to a phenotypic mitotic arrest. This movie corresponds to Figure 1D. Please click here to download this video.

Discussion

Identifying an Appropriate Cell

Key to imaging dividing S2 cells is first locating the proper cell. Time can be wasted imaging cells that are mistakenly thought to be ready to divide but fail to do so in a reasonable timeframe. Cells must be identified that have two distinct and visible centrosomes and an intact nucleus. Centrosomes must have microtubule fibers emanating from them, giving them a “star-like” appearance. An intact nucleus refracts light when adjusting focus, and also makes the approximate center of the cell darker in appearance. The nucleus will also contain the GFP:CID “dots”, another distinguishing feature to help in its identification. Cells with >2 centrosomes, tubulin puncta without tubulin fibers emanating from them, or cells with only one centrosome should be avoided. Additionally, if two centrosomes are visible, but a nucleus is not, NEBD has already occurred and the cell is too far advanced in its division to be imaged if a complete M-phase analysis is desired. Once an appropriate cell is found, speed in starting imaging is crucial as NEBD occurs quickly (typically within 3-5 min) and delaying the start of imaging can lead to missed opportunities. Focus the cell in the imaging software quickly such that both centrosomes are visible, or if centrosomes are out of plane with each other, set the focal point between the two, such that each can be captured when Z stacks are collected above and below the defined center point. Once focused, immediately set the offset between the stage and the live cell chamber surface using the infrared focus check (if available) and begin the imaging program. Although the protocol presented here is specifically tailored for experiments assessing NEBD-to-anaphase dynamics, simple modifications could suit readers studying alternative mitotic events. For NEBD-to-metaphase and anaphase-to-telophase imaging, we suggest 10 s image capture intervals as these processes are highly dynamic and occur quickly in S2 cells (5-10 min). Metaphase-to-anaphase transition (our typical experimental focus) is a longer process (20-30 min) in S2 cells, and we collect images at 30 s intervals. Lastly, telophase-through-cytokinesis occurs quite slowly in S2 cells, and we suggest 60 s intervals providing enough resolution for this approximate hour-long process.

Maintaining Focus Throughout the Imaging Program

If an infrared focus check device is not available, be sure to avoid bumping the microscope or table it sits upon, as this can result in the cell moving out of focus during the imaging program, leading to ambiguous, un-interpretable results. Pre-coating the live cell chamber wells with Poly-L-lysine can help in cell adherence, which can help avoid cell movement and is especially useful for setups without infrared focus checks. Additionally, setups including shock-absorbing platforms or air tables can further help avoid cells moving out of focus. Lastly, some software programs have pause functions allowing the user to refocus and resume the program.

Imaging Multiple Cells

Helpful to accumulation of data is that often multiple cells ready to divide can be placed within the same field of view for imaging. This can be especially useful for experiments in which cells have long M-phase durations or prolonged arrest (e.g., conditions that induce activation of the SAC). For these cells, we typically image until the cells are photobleached (approximately 2-3 h), but the full timing of arrested cells should be at the discretion of the researcher. Sometimes the multiple pre-dividing cells can be in different focal planes, in which case it may be useful to add z-stacks to accommodate all cells. Adding more z stacks can also help improve resolution. Caution should be taken in doing this, however, as it will expose cells to more radiation and lead to quicker photobleaching, as well as lead to large file sizes on hard disk storage devices.

Avoiding Photobleaching and Phototoxicity

Our method describes specific settings for exposure times, imaging intervals, z stacks, and percent transmittance for our LED light source that generally work for our experimental setup and desired outcomes. As microscopes and experimental goals may vary, what might work well for our system may lead to premature photobleaching or phototoxicity in others. Photodamage may present a lack of spindle movement, microtubule fragmentation, defective microtubule dynamics, and prolonged spindle checkpoint activation. One potential method to avoid such pitfalls is to limit the exposure time. Most modern cameras now possess wide dynamic ranges, and histograms can be adjusted to visualize structures even at very low exposures. Another technique is to increase the imaging interval (e.g., to several minutes between images) such that the number of times a cell is exposed to light over a total collection interval is reduced. This is advantageous particularly in imaging for longer periods of time (many hours), and can additionally help in decreasing the file size of such experiments. Limiting the number of z stacks taken can also help reduce light exposure, using 2 or even just 1 instead of 3. Further, adjusting the percent transmittance of light (for LED light sources), or utilizing neutral density (ND) filters (for both Halogen Lamp and LED light sources) will decrease the light intensity and coupled with longer exposure times can preserve visibility. By choosing cells with centrosomes already separated, overall exposure can be limited in that these cells usually enter mitosis quickly (within a minute or two) after beginning imaging. One can also limit the time that cells are allowed to be imaged before NEBD. Our lab typically sets this time at 10 min, but a more conservative limit can easily be imposed by the user. Additionally, a more ‘invasive’ measure we recommend is treatment with dsRNA targeted against a component of the SAC (such as Rod or Mad2). If an arrest phenotype is due to non-specific cell damage (e.g., defective microtubule dynamics), such a treatment is less likely to suppress the arrest compared to a bona fide effect of the gene of interest in the original experiment.

Future Directions

The method described here can be utilized on a relatively simple epifluorescent microscope to rapidly image live cell divisions and can be easily adapted to suit a researcher’s particular experimental design and goals. Several excellent methods for culturing, RNAi knockdown approaches, transient transfection, and fluorescence microscopy have been published for S2 and other Drosophila cells^{9,14,15,16,17}. Our protocol offers a couple of advantages. (1) The use of a double stable cell line (GFP:CID, mCherry:α-Tubulin) simultaneously marks two key mitotic structures, avoids the hassle and potential complications of transient transfection, and ensures that nearly all cells will express fluorescent markers as potential candidates for imaging. (2) The adaptation to mitosis adds to published protocols examining cytoskeletal dynamics in motile, non-dividing cells and extends to examining mitotic events in real time. While we believe ours is a powerful technique that adds to the repertoire of others, there can always be improvements made. One particular area of cell division that we have found hard to image is centrosome division. This occurs prior to NEBD and it is difficult to determine when a single centrosome is ready to separate into two. Using fluorescent cell cycle markers (Cyclin A and Cyclin B) could help resolve this problem, but comes at the expense of channels that could be used to visualize cell division components. Our best strategy for attempting to visualize centrosome division is to add multipoint acquisition to the imaging program (defining different points in the XY plane to image), but this can result in large files, and can also require (depending on the number of points) increasing the interval between image collection, decreasing time resolution of the resulting movie. Another solution could be synchronization of cells at a desired cell cycle stage using drugs that target cell cycle regulators followed by subsequent washout prior to imaging, although these methods may not be reliable in S2 cells specifically.

The protocol presented here provides a foundation for future applications in live cell imaging as well. With improved imaging and software technology, truly high throughput adaptations of this protocol using higher capacity, multi-chambered plates could be possible. Such innovations would make large-scale RNAi screens, such as those already achieved in fixed preparations^12,18, more tractable in a live cell format. Small molecule drug screens could also be envisioned as a means of identifying novel compounds targeting cell division processes. Improvement of fluorophores and optical filters could also lead to imaging of multiple mitotic components (not just the two we describe), enabling imaging of specific mitotic regulators and their interactions with DNA and/or the spindle. For example, generating cells with fluorescent reporters that express following the DNA damage or during apoptosis would be useful tools to identify novel genes involved in these processes. Similar approaches could be used to examine cell cycle dynamics using the expression of fluorescently tagged cyclins¹⁹.

Materials

Name	Company	Catalog Number	Comments
Bright-Line Hemacytometer	Sigma-Aldrich	Z359629-1EA	for cell counting
cellSens imaging software	Olympus
CELLSTAR Cell Culture Flask, 50 mL, 25 CM2, PS, Red Filter Screw Cap, Clear, Sterile, 10 PCS/BAG	Greiner Bio-One	690175
CELLSTAR Cell Culture Multiwell Plate, 6 well, PS, Clear, TC, Lid with condensation rings, sterile, single packed	Greiner Bio-One	657160
Centrifuge 5804 R	eppendorf	Cat. 022623508
Copper(II) sulfate pentahydrate, minimum 98%	Sigma-Aldrich	C3036-250G
Corning Fetal Bovine Serum	Fisher Scientific	MT35015CV
Effectene Transfection Reagent 1 mL	Qiagen	301425	for transient transfection
IX-83 Inverted Epifluorescent Microscope	Olympus
LabTek Chambered Slide Insert	Applied Scientific Instruments	I-3016
MEGAscript T7 Transcription Kit	Thermo Fisher Scientific	AM1334	For dsRNA production
MOXI Z Mini Automated Cell Counter Kit	ORFLO	MXZ001	for cell counting
MS-2000 XY Flat-Top Automated Stage and Controller	Applied Scientific Instruments
Nunc Lab-Tek II Chambered Coverglass (no 1.5 borosilicate glass) 8-well	Thermo Fisher Scientific	155409
Orca-Flash 4.0 LT Camera	Hammamatsu Photonics K.K.	C11440-42U
pMT/V5-His A Drosophila Expression Vector	Thermo Fisher Scientific	V412020
Poly-L-Lysine	Cultrex	3438-100-01
Purifier Logic+ Class II, Type A2 Biosafety Cabinets	Labconco	302310000
Schneider's insect medium	Sigma-Aldrich	S0146-100ML
Spectra Tub Centrifuge Tubes	VWR	470224-998
Uner Counter BOD Incubator	Sheldon manufacturing (VWR)	89409-346
X-CITE 120 LED	ExcelitasTechnologies		Led light source
Z -Drift Compensator (ZDC)	Olympus		infrared focus check