We are isolating and distributing deletion mutants to the community using a TMP-UV method (Gengyo-Ando and Mitani, 2000). To collect high-quality mutants, we used to examine the presence of wild-type DNA sequences when deletion sizes are longer than 1 kb. However, after a time-point, we changed the criteria shorter than before: 500 bp. When deletion sizes are shorter, we can easily identify such a rearrangement as we do “homo-hetero” tests (as seen by WT band in lane 1 in addition to internal band in lane 2 of type #2 mutant in Figure 1). Most of the mutants caused by rearrangements can be excluded this way and not posted publicly. We guess, however, there may be still a small number of such rearrangement artifacts in our mutant collections in earlier tm alleles (before tm2000). We re-examine the rearrangements when necessary and continuously improve the quality of the collection; we do the re-examination when we find strains that turned out to be homozygous viable, which were regarded as homozyogous inviable and did not examine the internal primer test before. We withdraw alleles when we find problems and try to replace them by isolating new alleles. We hope if people find such cases, please let us know.

Chemotaxis bioassays are necessary for identifying and characterizing signals that cause attraction or repulsion responses in C. elegans (Ward, 1973). One common method for performing bioassays is to paralyze worms with sodium azide once they reach the material and count the numbers in each area. Another is to record the entire bioassay, which allows you to extract additional data such as detailed spatial information (useful in lawn leaving assays), worm speed, and reversal frequency (Srinivasan et al., 2008; Buckingham and Sattelle, 2008; Bendesky et al., 2011). The downside to this method is its low throughput that is limited by the number of microscopes. We present here a multiple camera recording system using six webcams mounted above a custom made light box which allows the recording of six bioassays simultaneously (Fig. 1). These recordings are processed using a custom MATLAB script which outputs a histogram showing the positions of all worms over the course of the assay. Multiple histograms are summed together using image registration. Test assays were performed using diacetyl (a known attractant), verifying its usefulness in attraction bioassays (Fig. 2).

This apparatus is still under development, and we welcome suggestions from the community on how to improve it. Specifically, we have been unable to connect six cameras to one computer due to limitations in USB bandwidth and are using 2 computers instead. We would also like to be able to record assays with L4 and younger worms, which is currently difficult due to the low contrast of the videos. General methods are described below; details will be described in a further publication.

WormBox Construction

Six Microsoft LifeCam Cinema 720p HD webcams are mounted in a custom holder made of nylon above a PVC lightbox outfitted for brightfield microscopy. The reflector is white photo-quality paper, positioned at an approximately 45° angle. The light was constructed using 7 Philips Luxeon K2 Cool White LEDs. Four cameras are connected via USB 2.0 to a Windows 7 x64 computer with 16GB RAM and two cameras are connected to a Windows XP computer with 4GB RAM. The images from each camera are captured with the MATLAB Image Acquisition Toolbox and saved as often as possible (around 1 Hz).

Video Analysis

The background, which is calculated using principal component analysis, is subtracted from each image. The subtracted images are binarized and cleaned to remove stray pixels and connected components which are too small to be worms. The resultant series of binarized images are summed to generate a histogram, or a record of the locations of all worms across time. Multiple assays are aligned using the scoring regions as control points, and summed. All processing is done in MATLAB.

Acknowledgments

We thank Ryan Newsome and the Florida State University Machine Shop for help designing and for constructing the light box and the UF Center for Smell and Taste for machine shop funds (NIH P30 DC010364). We thank Chaevien Clendinen and Circe Lassegue for help with the project. This work was supported by 1R01GM085285 and 3R01GM085285-01A1S1.

We have developed a protocol that greatly speeds preparation of C. elegans samples for analysis by immunoblotting. A key point of simplification in the protocol is standardizing the age and numbers of nematodes used for the studies. Our immunoblot analyses use 15 to 50 age-synchronized nematodes per well of 10 or 15 lane mini protein gels (Invitrogen). The exact number varies depending on the nematode age, the antigen abundance and the quality of the antibody. However, to provide investigators a benchmark from which they can further optimize their studies, we provide three examples using Actin as the antigen and hermaphrodites as the source material: L4/35 nematodes, adult day1/30 nematodes, adult day3/20 nematodes. Use of a defined number of age synchronized nematodes enables us to load exactly the same amounts of total protein from multiple samples with very minimum variation between the gel wells.

The sample preparation does not require sonication or any other mechanical method to crush the nematodes. Synchronized adult animals first are cleaned by transferring to a NGM plate with no bacteria. Next, 15 to 50 nematodes are picked and placed into 20 μL of DDH₂O that is contained in the cup-like part of a severed Eppendorf tube lid (Fig.1). Once the nematodes are released in the water droplet, the lid is put on an intact Eppendorf tube containing 20 μL of 2X sample buffer already pipetted at the bottom of the tube (Fig. 1). The nematodes are spun into the 2X sample buffer (see below) for one minute at 12,000 RPM. The pelleted sample with nematodes is then flash frozen in an ethanol/dry ice bath; at this point the frozen sample can be stored in a -80⁰C freezer until one is ready to run the gel. The volumes used in the protocol can be adjusted to match the sample size (for example, 10μL H₂O+10 μL 2X sample buffer for 15 adult day1 nematodes).

Prior to running the samples in a mini gel, the samples are incubated at 95⁰C in a heating block for five minutes, followed by five minutes of cooling at room temperature. The samples are then heated again for the second time at 95⁰C for five minutes and can be loaded onto a gel. The subsequent immunoblot protocol varies depending on the particular antigen and antibody used. In our laboratory, proteins in the gels are transferred to a PVDF (BioRad Inc.) filter using NuPage transfer buffer with 10% Methanol and 0.01% SDS at 20 volts overnight or a shorter period (3 to 5 hrs) until no marker protein is left in the gel. Incubation with the primary antibody is commonly performed in PBS containing 4% blotting grade milk with 0.025% Tween-20 for two to twenty four hours in a cold room (4⁰C), depending on the antigen affinity of the antibody. Incubation with the secondary antibodies is done for two hours at room temperature with three subsequent washes (5 min X 3) with PBS containing Tween-20 prior to treating with ECL solution.

2X Sample buffer: 100mM Na-Tricine pH 7.8, 100mM DTT, 14% W/V Glycerol, 4% LDS, 0.05% CHAPS and 0.002% Bromphenol Blue (can be added later if protein concentration is to be determined for the lysates). The sample buffer can be frozen in aliquots in -20⁰C for long-term storage.

2μL HALT (Protease inhibitors cocktail, Sigma) should be added to 98μL 2X Sample buffer prior to mixing with nematodes.