Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.
The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.
Caenorhabditis elegans are commonly used in aging studies because of their short generation time (approximately 3 days), short lifespan (approximately 3 weeks), ease of cultivation in the laboratory, high degree of evolutionary conservation of molecular processes and pathways with mammals, and wide availability of genetic manipulation techniques. In the context of aging studies, C. elegans allow for the rapid generation of longevity data and aged populations for the analysis of late-life phenotypes in live animals. The typical approach for conducting worm aging studies involves manually measuring the lifespan of a population of worms maintained in groups of 20 to 70 animals on solid agar nematode growth media (NGM) in 6 cm Petri plates1. Using age-synchronized populations allows the measurement of lifespan or cross-sectional phenotypes in individual animals across the population, but this method precludes monitoring the characteristics of individual animals over time. This approach is also labor-intensive, thus restricting the size of the population that can be tested.
There are a limited number of culture methods that allow for the longitudinal monitoring of individual C. elegans throughout their lifespan, and each has a distinct set of advantages and disadvantages. Microfluidics devices, including WormFarm2, NemaLife3, and the "behavior" chip4, among others5,6,7, allow the monitoring of individual animals over time. Culturing worms in liquid culture using multi-well plates similarly allows the monitoring of either individual animals or small populations of C. elegans over time8,9. The liquid environment represents a distinct environmental context from the common culture environment on solid media in Petri plates, which can alter aspects of animal physiology that are relevant to aging, including fat content and the expression of stress-response genes10,11. The ability to directly compare these studies to the majority of data collected on aging C. elegans is limited by differences in potentially important environmental variables. The Worm Corral12 is one approach developed to house individual animals in an environment that more closely replicates typical solid media culture. The Worm Corral contains a sealed chamber for each animal on a microscope slide using hydrogel, allowing the longitudinal monitoring of isolated animals. This method uses standard brightfield imaging to record morphological data, such as body size and activity. However, animals are placed in the hydrogel environment as embryos, where they remain undisturbed throughout their lifespan. This requires the use of conditionally sterile mutant or transgenic genetic backgrounds, which limits both the capacity for genetic screening, as each novel mutation or transgene needs to be crossed into a background with conditional sterility, and the capacity for drug screening, as treatments can only be applied once to the animals as embryos.
An alternative method developed by the Fang-Yen lab allows the cultivation of worms on solid media in individual wells of a microfabricated polydimethylsiloxane (PDMS) device called a WorMotel13,14. Each device is placed into a single-well tray (i.e., with the same dimensions as a 96-well plate) and has 240 wells separated by a moat filled with an aversive solution to prevent the worms from traveling between wells. Each well can house a single worm for the duration of its lifespan. The device is surrounded by water-absorbing polyacrylamide gel pellets (referred to as "water crystals"), and the tray is sealed with Parafilm laboratory film to maintain the humidity and minimize the desiccation of the media. This system allows healthspan and lifespan data to be gathered for individual animals, while the use of solid media better recapitulates the environment experienced by animals in the vast majority of published C. elegans lifespan studies, thus allowing more direct comparisons. Recently, a similar technique has been developed using polystyrene microtrays that were originally used for microcytotoxicity assays15 in place of the PDMS device16. The microtray method allows for the collection of individualized data for worms cultured on solid media and has improved capacity for containing worms under conditions that would typically cause fleeing (e.g., stressors or dietary restriction), with the trade-off being that each microtray can only contain 96 animals16, whereas the multi-well device utilized here can contain up to 240 animals.
Presented here is a detailed protocol for preparing multi-well devices that is optimized for plate-to-plate consistency and the preparation of multiple devices in parallel. This protocol was adapted from the original protocol from the Fang-Yen laboratory13. Specifically, there are descriptions for techniques to minimize contamination, optimize the consistent drying of both the solid media and the bacterial food source, and deliver RNAi and drugs. This system can be used to track individual healthspan, lifespan, and other phenotypes, such as body size and shape. These multi-well devices are compatible with existing high-throughput systems to measure lifespan, which can remove much of the manual labor involved in traditional lifespan experiments and provide the opportunity for automated, direct longevity measurement and health tracking in individual C. elegans at scale.
1. Preparation of stock solutions and media
NOTE: Before beginning the preparation of the multi-well devices, prepare the following stock solutions and media.
2. Printing the 3D multi-well device mold
NOTE: Each device is molded from PDMS using a custom 3D-printed mold. A single mold can produce as many devices as needed; however, if attempting to prepare multiple devices at the same time, one 3D-printed mold is required for each device to be made in parallel.
3. Preparation of the multi-well device
NOTE: This section describes how the 3D-printed mold is used to create the PDMS multi-well device.
4. Streaking the bacteria
NOTE: Begin preparing the bacteria that will be used as the worms' food source while they are on the multi-well device. The most common bacteria is Escherichia coli strain OP50 (or strain HT115 for RNAi experiments). Complete this step at least 2 days prior to adding the worms to the device.
5. Preparation of the multi-well device for media loading
NOTE: The surface of the silicone PDMS material that makes up the device is hydrophobic, which prevents the small-volume wells and aversive moats from being filled with NGM and copper sulfate, respectively. To circumvent this problem, an oxygen plasma is used to temporarily modify the surface properties of the device to be hydrophilic, allowing the wells and moat to be filled within a limited time window (up to ~2 h). This section lays out the steps for completing the plasma-cleaning process. Complete this step at least 1 day before spotting the device wells with bacteria, as lingering effects of the plasma clean can interfere with spotting. Given the timing of sections 5-7, the practical limit for these steps per technician is three devices in parallel.
6. Filling the wells with lmNGM
NOTE: A dry bead bath incubator should be on and preheated from step 5.1. Ensure that the bath has reached 90 °C.
7. Adding copper sulfate to the moat
NOTE: This device's wells are surrounded by a continuous moat. Here, the moat is filled with copper sulfate, which acts as a repellent and deters the worms from fleeing from their wells.
8. Adding autoclaved water crystals
NOTE: To maintain humidity within the plate and prevent desiccation of the lmNGM, each device is surrounded by saturated water-absorbing polyacrylamide crystals.
9. Preparation of an age-synchronized population of worms
NOTE: The following steps yield a synchronized population of worms that are ready to add to the multi-well device at the fourth larval stage (L4). However, worms at different stages of development can also be added. This step should be completed 2 days before adding the worms to the device if L4s are desired. Adjust the timing of synchronization for the desired life stage.
10. Inoculating the bacterial culture
NOTE: Bacteria are used as the primary food source for C. elegans, most commonly E. coli strains OP50 or HT115. The bacteria are concentrated 10-fold, which should be accounted for in the volume of the prepared culture. Prepare a bacterial culture the day before spotting the device.
11. Spotting the wells with concentrated bacteria
NOTE: A small volume of concentrated bacteria is added to each well, which is sufficient to feed the worms for their entire lifespan on the device. The bacterial culture needs to be dried before the worms can be added to the wells. As the media volume in each well is small (14-15 µL) relative to the bacteria volume added (5 µL), the chemical content of the bacterial media can impact the chemical environment of the well. To account for this, the bacteria are concentrated and resuspended in salt water to remove depleted LB while avoiding hypoosmotic stress. There is no salt added to the lmNGM recipe (see steps 1.3-1.4) as it is added at this stage.
12. Adding worms to the multi-well device
13. Finishing the preparation of the device for long-term use
NOTE: These steps ensure that the device wells remain hydrated for the duration of the experiment.
14. Collection of the data
NOTE: The purpose of this study is to describe the culture methodology. Once populated, multi-well devices are compatible with the longitudinal monitoring of a variety of phenotypes. Here, basic guidance for measuring several of the most common parameters is provided.
15. Reusing the devices
NOTE: After an experiment is complete, the multi-well devices can be cleaned and reused up to three times. Additional reuse begins to impact the worm phenotypes, possibly caused by chemicals from the media or bacteria building up in the walls of the PDMS material.
The WorMotel culture system can be used to gather a variety of data, including regarding lifespan, healthspan, and activity. Published studies have utilized multi-well devices to study lifespan and healthspan13,14, quiescence and sleep22,23,24, and behavior25. Lifespan can be scored manually or through a collection of images and downstream imaging analysis. In the former approach, the worms can be manually observed following a stimulus (e.g., tapping the plate or exposure to blue light) every 1-3 days and scored as dead if no movement is observed, similar to standard methods on Petri plates1. The latter approach is similar, except that worm movement can be determined by comparing frame-to-frame differences between the images taken after the stimulus has been applied. This provides an added benefit in that movement provides information both on the activity level of individual animals at that time point and provides a metric by which lifespan (e.g., cessation of movement) and healthspan (multiple definitions have been proposed) can be determined. The images can further be used to extract additional physiological parameters such as body size, body shape, and body posture.
To demonstrate the capacity of the system, we examined the classical epistatic relationship between the insulin receptor, encoded by the gene daf-2, and the downstream FOXO family transcription factor encoded by daf-16 in the context of lifespan, healthspan, and daily activity for individual animals. Wild-type (strain N2) and daf-16(mu68) loss-of-function (strain CF1038) C. elegans fed E. coli (strain HT115) expressing either control (empty vector; EV) or daf-2 RNAi feeding constructs were cultured in multi-well devices, and each animal was monitored for lifespan (Figure 2A), healthspan (Figure 2B), and daily activity (Figure 2C). The activity was monitored daily by taking a series of still images every 5 s for 2 min, with the worms being exposed to bright blue light for 5 s at 1 min to stimulate activity (as per Churgin et al.13). The daily activity for each animal was estimated by normalizing the background across wells and images, identifying the worm area in each image, and calculating the change in area between adjacent images. Lifespan was defined as the age at which activity was last observed for each worm, and healthspan was defined as the age at which a worm could no longer move a full body length. As expected from numerous previous studies (e.g., Kenyon et al.26, Murphy et al.27), the daf-16(mu86) mutation resulted in short lifespans and prevented lifespan extension from the RNAi knockdown of daf-2 (Figure 2A). A similar pattern was observed for healthspan (Figure 2B). As an advantage of using multi-well device culture systems, the capacity to track individual animals throughout life allows a detailed analysis of the individual variation in each measured phenotype across the population. For instance, the variation in lifespan and healthspan across individual animals can be compared in either absolute (Figure 2D) terms or as a fraction of the total lifespan (Figure 2E). Early-life phenotypes can further be compared to late-life phenotypes, including lifespan, in individual animals across a population. For instance, the cumulative activity for each individual animal across the lifespan (i.e., the area under the curve [AUC] for individual activity) correlated better with lifespan (Figure 2F) than cumulative lifespan up to day 5 of life (Figure 2G) across all the conditions measured. We emphasize that the purpose of this work is to provide a detailed protocol for constructing the multi-well environment for tracking individual animals over time, not for measuring a specific phenotype using the device. The representative results presented in Figure 2 provide just one example of the phenotypes that can be measured in this system. Once constructed, the multi-well environment is compatible with a wide range of techniques for measuring the phenotypes of free-crawling worms on solid media.
Figure 1: Schematic of the microfabricated multi-well devices. (A) Individual C. elegans are cultured on solid low-melt nematode growth media (lmNGM) agarose pads seeded with bacterial food in individual wells. The space between the wells is coated with an aversive chemical (copper sulfate) to isolate each worm within its well. Each device is secured inside a single-well tray. The perimeter of the tray is filled with water crystals to maintain the humidity. The tray is sealed with Parafilm to allow oxygen exchange. Image created with BioRender.com. (B) Overview of the multi-well device indicating the suggested order for loading the wells. The inner wells (white) receive 14 µL of lmNGM. The outer wells (gray) receive 15 µL of lmNGM. Please click here to view a larger version of this figure.
Figure 2: Correlation of measured phenotypes across populations in individual animals using multi-well devices. All the panels provide data from the same experiment comparing four groups of animals: wild-type (N2) animals subject to empty vector EV(RNAi) (N = 138), wild-type animals subject to daf-2(RNAi) (N = 151), daf-16(mu86) animals subject to EV(RNAi) (N = 123), and daf-16(mu86) animals subject to daf-2(RNAi) (N = 135). (A) The lifespan extension from daf-2(RNAi) is blocked by the daf-16(mu86) null mutation. Pairwise significance between groups determined by the log-rank test (survdiff function in R). (B) The healthspan-defined here as the day at which an animal can no longer move a full body length-extension from daf-2(RNAi) is blocked by the daf-16(mu86) null mutation. Pairwise significance between groups determined by the log-rank test (survdiff function in R). (C) The 3 day rolling mean of activity across the lifespan is reduced by both daf-16(mu86) and daf-2(RNAi). Significance calculated by the Mann-Whitney U test to compare the area under the curve for activity across the lifespan for individual animals between groups. (D) Healthspan and lifespan for each population as absolute values (mean ± standard error of the mean). (E) Healthspan and lifespan for each population normalized to total lifespan within each group (mean ± standard error of the mean). (F) The cumulative activity across the lifespan (area under the curve [AUC] across the lifespan) for individual animals correlates better with lifespan than (G) the activity for individual animals at any specific day across the lifespan (the activity correlation on day 8, representing the point at which the mean activity is maximized, is shown), as calculated by linear regression (lm function in R). n.s. = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. All p-values were adjusted for multiple comparisons using the Bonferroni method for comparisons made within each panel. Please click here to view a larger version of this figure.
Supplementary File 1: STL file for printing the 3D multi-well device mold Please click here to download this File.
The WorMotel system is a powerful tool for gathering individualized data for hundreds of isolated C. elegans over time. Following the earlier studies using multi-well devices for applications in developmental quiescence, locomotory behavior, and aging, the goal of this work was to optimize the preparation of multi-well devices for the long-term monitoring of activity, health, and lifespan in a higher-throughput manner. This work provides a detailed protocol for preparing multi-well devices that optimizes many of the steps from the original protocol13, highlights key points that may present technical difficulties, and provides a discussion about reusing the plates and other materials.
For the purposes of scaling-as a lab that currently prepares between 10 and 20 devices in a typical week-a top consideration was whether the devices could be reused and, if so, to what degree. There is a higher cost in terms of both time and money in preparing PDMS devices relative to conducting traditional culture on Petri plates, but these higher costs can be reduced by reusing the PDMS or other components of the system. With many reuses, the PDMS began to develop a yellow coloration, likely reflecting the accumulation of compounds from the lmNGM media or bacteria. The animals cultured on these plates also displayed a higher rate of fleeing and reduced lifespans. Based on dozens of experiments, three uses are optimal for reusing these PDMS devices, allowing age-related phenotypes to be assessed without a measurable impact from PDMS degradation while reducing the number of new devices that need to be molded (thus saving on costs). We further confirmed that experiment-matched animals grown on devices on their first, second, and third use produced survival curves that were nearly indistinguishable and not statistically different (data not shown). The trays used to contain the devices are made of polystyrene and can be cleaned and reused indefinitely if they remain free of scratches or other marks that could interfere with visualizing the worms.
A key challenge for preparing multi-well devices for applications that last more than ~2 weeks is the prevention of plate contamination by environmental bacteria and fungus. There are multiple steps at which sterilization is critical for preventing contamination. These include autoclaving all the devices prior to use, boiling the devices intended for reuse, autoclaving the absorbent water crystals used to maintain humidity, cleaning the trays that contain the devices with both bleach and ethanol before use, and filter-sterilizing the detergent solution that is applied to the lid of the tray to prevent fogging and added to the copper sulfate solution. Implementing each of these changes notably reduced contamination events, allowing the sealed devices to be consistently used for longitudinal monitoring across the lifespan, even under pro-longevity conditions (e.g., knockout of daf-2) that require monitoring for >45 days. The protocol described here includes two modifications to the original protocol for long-term applications designed to maintain consistent well drying and prevent desiccation. First, the overall depth of the device was increased by 2 mm to increase the capacity for water crystals. Over long experiments, particularly in a low-humidity environment, too few water crystals in the tray led to the desiccation of the lmNGM. Along with more water crystals, it was necessary to increase the volume of agarose that was added to the wells along the edge of the device. These wells tended to dry first following the well loading (section 6) and shrink. Using 14 µL of agarose on the inside wells was enough volume to fill the wells completely without creating the domed top that results from over-filling the wells. Adding 15 µL of agarose to the outer wells provided enough volume that, when the wells began drying out, they shrank to a level comparable to the 14 µL added in the inner wells.
One of the largest deviations from the original protocol was to reverse the order in which the user loads the lmNGM (section 6) and copper sulfate (section 7). Originally, the copper sulfate was added to the moat first, followed by filling the wells with lmNGM13. It was observed that filling the wells with lmNGM as soon after plasma cleaning as possible improved the adherence of the lmNGM to the well walls. Waiting too long after the plasma cleaning resulted in wells with bubbles and domed tops, which can interfere with visualizing the worms. Prioritizing filling the wells over adding the copper sulfate is particularly important when preparing multiple devices at the same time to ensure consistent, high-quality lmNGM surfaces. A downside of filling the wells first is that the hydrophilic surface modification produced by the plasma cleaner will have worn off noticeably by the time the user moves on to adding the copper sulfate. Copper sulfate does not flow easily through the moat when the surface becomes less hydrophilic, thus making it challenging to achieve complete coverage. Adding detergent to the copper sulfate solution to act as a surfactant improves the flow of the solution through the moat. A platinum wire pick can also be used to gently guide the copper sulfate through the moat by breaking the tension at any points where the copper sulfate is having difficulty flowing. Furthermore, if the copper sulfate solution were left in the moat, it would easily spill into the wells and contaminate the lmNGM surface when tilting the tray. The nature of the loading process makes it nearly impossible to keep the device sufficiently level throughout to prevent copper from contaminating a subset of the wells. To account for this, the copper sulfate solution is removed from the moat (step 7.3), and the residual copper sulfate left behind is sufficient to deter most worms from fleeing. As a final note on the use of copper sulfate as an aversive barrier, some repellents can affect age-associated phenotypes, including lifespan. The use of copper sulfate in these multi-well devices was examined by Churgin et al.13 and found to have no detectable impact on either lifespan or development.
Other minor updates to the protocol focus on improving the steps taken to prepare the PDMS. An extra degassing step after mixing the PDMS base and curing agent was added, as this is a process that generates many bubbles. Removing the majority of the bubbles before adding the mixture to the device molds minimizes the bubbles remaining after the second degassing step. To ensure that the bottom of the device is entirely flat and even, a feature that is important for whole-plate imaging, a piece of glass or acrylic was laid across the top of the filled mold. This step is not essential-though still helpful-for applications that only examine one well at a time, as the user can manually adjust the focus for each well. Finally, curing the PDMS at a higher temperature (55 °C) was necessary at the location where this version of the protocol was optimized (Tucson, Arizona, USA), in contrast to the 40 °C indicated by the original protocol (optimized in Philadelphia, Pennsylvania, USA). This suggests that differences between locations (such as climate or the precise reagents and equipment used) can affect the specific steps in the protocol, such as the curing temperature or drying techniques, and that these may need to be optimized at each site. For instance, environmental humidity plays a large role in determining the drying time for the plates after spotting, and this can vary greatly between locations or seasons.
In principle, this multi-well device system can be used to collect data on any phenotype that can be measured under standard brightfield microscopy on Petri plates-lifespan, healthspan, unstimulated/stimulated movement, body size and shape, movement geometry-with the added capability of tracking those metrics for individual worms over time. As noted in the introduction, there are other methods for acquiring individual whole-lifespan data. Microfluidics devices28 or multi-well plates29 offer the capability to follow the life history of individual animals, but only by culturing the worms in liquid media. A liquid environment can alter the worms’ transcriptome10,11,30 relative to solid media and induces distinct physiological and behavior changes, including episodic swimming31, increased energy expenditure10,32, and elevated oxidative stress10. The degree to which lifespan and other metrics related to healthy aging can be directly compared between liquid and solid media is unclear. Solid culture multi-well devices allow single worm tracking in an environment that is comparable to standard group culture on Petri plates. The curved structure of the device wall allows for imaging of worms anywhere inside the well, making these devices compatible, in principle, with more sophisticated tools for single-worm analysis, such as Worm Tracker33.
The ability to monitor individual animals throughout the course of an experiment on a multi-well device is accompanied by several limitations. First, even with an optimized protocol, these devices require more time to set up per animal than standard culture on Petri plates, thus providing single-animal data at the cost of overall sample size. However, the worms are also isolated to individualized wells, removing some of the complications associated with automated lifespan measurement, such as automatically detecting individual animals that stop moving while touching one another. Automation more than recaptures the time lost to the more complex setup and provides the opportunity for more high-throughput screening13,18. Second, experimental conditions that the worms dislike more than the copper sulfate moat, such as strong stress-inducing agents (e.g., paraquat) or dietary restriction, will lead to the worms fleeing from the wells and into the moat. Third, while the PDMS devices allow brightfield and darkfield microscopy, the PDMS material tends to have both high and uneven background fluorescence, the latter likely resulting from dust or other microparticles becoming embedded in the PDMS during molding, which limits their application in fluorescent microscopy. Finally, the discoloration observed in the PDMS for devices reused over multiple experiments and the associated reduction in lifespan and increase in fleeing suggests that the media components and other added chemicals leech into the PDMS over time. The degree to which this may impact aging phenotypes or drug treatments is currently being explored. As mentioned previously, there is an alternative method for a single-worm culture that is similar to PDMS multi-well devices but instead uses commercially available microtrays to culture isolated animals16. These microtrays are made from polystyrene, avoiding the potential issue of leeching media components, and have consistent fluorescence background, allowing for direct in vivo fluorescence imaging directly on the plate. The microtray wells are also surrounded by palmitic acid in place of the copper sulfate used in the protocol described here, which is more aversive and reduces the fraction of worms that leave their wells, even in the case of stress-inducing agents and dietary restriction. These benefits are realized at the cost of lower packing efficiency of the wells, as the microtrays allow a maximum of 96 worms to be cultured in a single tray in contrast to the 240 worm capacity of the PDMS devices. The specifics of the desired outcome of an experiment will influence which of these systems should be utilized.
The authors have nothing to disclose.
This work was supported by NIH R35GM133588 to G.L.S., a United States National Academy of Medicine Catalyst Award to G.L.S., the State of Arizona Technology and Research Initiative Fund administered by the Arizona Board of Regents, and the Ellison Medical Foundation.
2.5 lb weight | CAP Barbell | RP-002.5 | |
Acrylic sheets (6 in x 4 in x 3/8 in) | Falken Design | ACRYLIC-CL-3-8/1224 | Large sheet cut to smaller sizes |
Ampicillin sodium salt | Sigma-Aldrich | A9518 | |
Autoclavable squeeze bottle | Nalgene | 2405-0500 | |
Bacto agar | BD Difco | 214030 | |
Bacto peptone | Thermo Scientific | 211677 | |
Basin, 25 mL | VWR | 89094-664 | Disposable pipette basin |
Cabinet style vacuum desiccator | SP Bel-Art | F42400-4001 | Do not need to use dessicant, only using as a vacuum chamber. |
CaCl2 | Acros Organics | 349615000 | |
Caenorhabditis elegans N2 | Caenorhabditis Genetics Center (CGC) | N2 | Wildtype strain |
Carbenicillin | GoldBio | C-103-25 | |
Centrifuge | Beckman | 360902 | |
Cholesterol | ICN Biomedicals Inc | 101380 | |
Compressed oxygen tank | Airgas | UN1072 | |
CuSO4 | Fisher Chemical | C493-500 | |
Dry bead bath incubator | Fisher Scientific | 11-718-2 | |
Escherichia coli OP50 | Caenorhabditis Genetics Center (CGC) | OP50 | Standard labratory food for C. elegans |
Ethanol | Millipore | ex0276-4 | |
Floxuridine | Research Products International | F10705-1.0 | |
Hybridization oven | Techne | 731-0177 | Used to cure PDMS mixture, any similar oven will suffice |
Incubators | Shel Lab | 2020 | 20 °C incubator for maintaining worm strains and 37 °C incubator to grow bacteria |
Isopropyl ß-D-1-thiogalactopyranoside (IPTG) | GoldBio | I2481C100 | |
K2HPO4 | Fisher Chemical | P288-500 | |
KH2PO4 | Fisher Chemical | P286-1 | |
Kimwipes | KimTech | 34155 | Task wipes |
LB Broth, Lennox | BD Difco | 240230 | |
Low melt agarose | Research Products International | A20070-250.0 | |
MgSO4 | Fisher Chemical | M-8900 | |
Microwave | Sharp | R-530DK | |
Multichannel repeat pipette, 20–200 µL LTS EDP3 | Rainin | 17013800 | The exact model used is no longer sold, a similar model's catalog number has been provided |
NaCl | Fisher Bioreagents | BP358-1 | |
Nunc OmniTray | Thermo Scientific | 264728 | Clear polystyrene trays |
Parafilm M | Fisher Scientific | 13-374-10 | Double-wide (4 in) |
Petri plate, 100 mM | VWR | 25384-342 | |
Petri plate, 60 mM | Fisher Scientific | FB0875713A | |
Plasma cleaner | Plasma Etch, Inc. | PE-50 | |
PLATINUM vacuum pump | JB Industries | DV-142N | |
PolyJet 3D printer | Stratasys | Objet500 Connex3 | PolyJet 3D printing services provided by ProtoCAM (Matrial: Vero Rigid; Finish: Matte; Color: Gloss; Resolution: X-axis: 600 dpi, Y-axis: 600 dpi, Z-axis: 1600 dpi) |
Shaking incubator | Lab-Line | 3526CC | |
smartSpatula | LevGo, Inc. | 17211 | Disposable spatula |
Superabsorbent polymer (AgSAP Type S) | M2 Polymer Technologies | Type S | Referred to in main text as "water crystals" |
SYLGARD 184 Silicone Elastomer base | The Dow Chemical Company | 2065622 | |
SYLGARD 184 Silicone Elastomer curing agent | The Dow Chemical Company | 2085925 | |
Syringe filter (0.22 µm) | Nest Scientific USA Inc. | 380111 | |
Syringe, 10 mL | Fisher Scientific | 14955453 | |
TWEEN 20 | Thermo Scientific | J20605-AP | Detergent |
Vacuum pump oil | VWR | 54996-082 | |
VeroBlackPlus | Stratasys | RGD875 | Rigid 3D printing filament |
Weigh boat | Thermo Scientific | WB30304 | Large enough for PDMS mixture volume |