This work has been funded by NSF grant IOB#0615996.

Part 1: Preparing nematode growth media (NGM) Petri plates
Experimental nematodes such as C. elegans and P. pacificus are typically cultured in the laboratory on 6 cm Petri plates containing Nematode Growth Medium (NGM) 1. Worms are kept at 20&deg;C, but can be incubated at a range of temperatures (between 4&deg;C - 25&deg;C), depending on the nematode species and experimental design. To prepare plates with NGM, standard sterile techniques have to be used to prevent fungal and bacterial contamination. Following is the protocol to prepare NGM plates:
<ol>
	<li>Weigh 15 g agar, 2.4 g NaCl, 2 g Tryptone, and 2.72 g KH2PO4 in 2 L conical flask, make volume up to 1 L with water, autoclave and allow it to cool to 60&deg; C in a water bath.</li>
	<li>Add 0.8 ml each of 1 M CaCl2, cholesterol (5 mg/ml in ethanol), and 1 M MgSO4. To prevent fungal and bacterial contamination, 1 ml streptomycin (100 mg/ml) and 1ml nystatin (10 mg/ml) can be added to each liter of medium.</li>
	<li>Using a Unispense machine and maintaining sterile conditions, pour the media into the desired plates. The plates should be filled to about 2/3rd full. A standard 60 mm diameter plate requires about 10 ml of the media. Do not move the plates until the agar is completely gelified; otherwise there will be an uneven surface of agar, making it difficult to focus worms on the stereomicroscope.</li>
	<li>Once dried, the plates can be seeded immediately or stored at 4&deg; C until seeding with bacteria.</li>
</ol>
Part 2: Preparing OP50 bacteria and seeding NGM Petri plates
P. pacificus is cultured in the laboratory by feeding on the E.coli OP50 strain. OP50 has a limited growth on NGM plates, thereby forming a thin lawn that facilitates the visualization of the worms1. To grow a culture of OP50, use LB-broth:
<ol>
	<li>Add 5 g bactotryptone, 2.5 g yeast extract and 2.5 g NaCl to a 500 ml screw cap bottle, make up the volume to 500 ml with distilled water and autoclave.</li>
	<li>Inoculate the broth with a single colony of OP50 from the culture plate under sterile conditions and let it grow overnight at 37&deg; C.</li>
	<li>LB-OP50 is ready to be used and can be stored at 4&deg; C for several months if sterility is maintained. Whenever plates need to be seeded, pour a small amount of the broth into a small sterile beaker and this will help prevent contamination of the stock culture.</li>
	<li>To seed plates, dispense 100-120 &mu;l of the OP50 onto the 60 mm diameter NGM Petri plate and swirl to spread it.</li>
	<li>Incubate these seeded plates overnight at room temperature for the lawn to grow. These seeded plates can now be stored at 4&deg; C for up to 3 weeks. Storing them in an inverted position helps prevent rapid drying.</li>
</ol>
Part 3: Making a pick
In this section we describe how to make a pick for worms. The pick is used to transfer worms from one location to another. The pick is made of 30 gauge 90% platinum 10% iridium wire, though some researchers may prefer slightly different metal compositions:
<ol>
	<li>Cut about 3-4 cm of wire, place 0.5 cm of it inside the tip of a Pasteur pipette (whose tip was broken to a smaller length), and then seal it into the glass over a Bunsen burner. The length of the wire protruding from the glass is about 3-3.5 cm, but can vary according to individual preferences.</li>
	<li>Flatten the end of the wire that is protruding using a hammer or pliers. Then bend the flattened portion upwards to form a scoop.</li>
	<li>Smoothen the sharp edges of the pick with a sand paper to prevent damaging the worm or the agar.</li>
</ol>
Part 4: Picking worms
<ol>
	<li>To pick worms, sterilize the platinum wire on a flame and drag the flattened tip of the pick along the bacterial lawn to coat the tip with thick sticky bacteria, taking care not to puncture or damage the agar surface.</li>
	<li>Using the stereomicroscope, very lightly brush the sticky tip against the worm/worms to be picked till the worm sticks on to the bacteria on the pick.</li>
	<li>Once the worm is on the tip of the pick, transfer it immediately to the new plate by touching or sliding the tip of the pick against the bacterial lawn of the new plate, and the worm should crawl off. Care must be taken not to damage the surface of agar on the plate else the worms crawl into the holes making it difficult to retrieve them. If the worm stays on the pick for too long, it might desiccate.</li>
</ol>
Part 5: EMS mutagenesis
Ethyl methane sulfonate (EMS) induces a broad spectrum of mutations in the in the genome 2. Mutants can be identified by defects like egg laying, muscle defects, sex determination, dauer formation, behavior, and gonad formation. The protocol for EMS mutagenesis is similar to that described for C. elegans 1.
<ol>
	<li>Prepare 500 ml (or more) of 1N NaOH.</li>
	<li>Take 4-5, 6 cm diameter Petri plates full of worms, containing a few hundred worms at the third larval juvenile (J3) stage and wash the worms off using 2-3 ml of sterile M9 per plate.</li>
	<li>Collect all the worms in a sterile, disposable 15 ml centrifuge tube. Centrifuge at 1500 x g for 5-7 minutes or until the worms form a pellet.</li>
	<li>Aspirate the liquid and use 2 ml of M9 to re-suspend the worms. Add 20 &mu;l EMS to a tube containing 2ml of M9 and shake to dissolve it. Then add this EMS solution to the 2ml of worm suspension and swirl gently. The final concentration of EMS will be 47 mM. Caution: EMS is a very strong mutagen and should be handled in a fume hood. All materials (gloves, pipettes, tips) that come in contact with this mutagen should be treated with 1N NaOH to inactive the EMS. Wear double gloves during the entire EMS mutagenesis procedure. Dispose the hazardous waste according to your institution's guidelines.</li>
	<li>Place the centrifuge tube and cover the cap with parafilm; place it horizontally in the fume hood and let the worms incubate for 3.5 hours. The tube can also be placed on a low speed rocker.</li>
	<li>Turn the tube and cover the cap with parafilm; place in vertical position and incubate until the worms sink.</li>
	<li>Aspirate the supernatant and discard it into a beaker/flask containing 1N NaOH. This will inactivate the EMS.</li>
	<li>Add 5 ml M9 to the worms, invert the tube 25-30 times to wash the worms, centrifuge at 1500 x g for 5-7 mins or until the worms form a pellet. Aspirate the supernatant and discard into the 1N NaOH beaker.</li>
	<li>Repeat the washing step at least 3 times.</li>
	<li>After the final wash, re-suspend the worm pellet in minimal amount (~500 &mu;l) of M9 and pipette them out onto NGM plates containing a lawn of OP50 bacteria. At this point you no longer need to work in the chemical hood.</li>
	<li>Let the liquid dry for a few minutes and then pick and transfer healthy looking J4 larvae to fresh seeded plates. Discard the original plate according to your institution's hazardous waste guidelines.</li>
	<li>Let the mutagenized worms self fertilize. Using a stereomicroscope, screen the F2 generation phenotypes for recessive mutants of interest.</li>
</ol>
Part 6: Psoralen mutagenesis
In this section we describe the procedure for mutagenesis using TMP in conjunction with long wavelength ultraviolet (UV). TMP/UV method of mutagenesis is widely used to induce deletion mutations within a genome 3.
<ol>
	<li>Collect worms from 4-5, 6 cm diameter Petri plates into a 15 ml centrifuge tube as described in the previous procedure.</li>
	<li>Add 10 ml M9, invert 20 times to wash the worms, and centrifuge at 1500 x g for 5 minutes. Discard the supernatant and repeat the washing for a total of 3 times. Aspirate the liquid and re-suspend the worms in about 10 times their volume of M9 containing 30 &mu;g/ml of TMP. Pipette 20 &mu;l of TMP (3 mg/ml) into 2 ml of M9. Like EMS, TMP is a potent carcinogen and must be handled taking proper safety measures. To prevent from potential risk of the UV light proper eye wears should be used. The disposables must be discarded in proper biohazard bags.</li>
	<li>Incubate the tube in the dark for 15 minutes at room temperature on a low speed rocker (40 UPM). Set the covered tube vertically for 10 minutes to let all the worms sink.</li>
	<li>Remove the worms from the bottom of the tube with a Pasteur pipette and transfer them onto an unseeded NGM plate. Discard the supernatant following your institution's hazardous chemical guidelines.</li>
	<li>Keep the plate in dark until the excess TMP is absorbed into the plate.</li>
	<li>Using a UV intensity meter, set the distance between the long wave UV source and the plate such that the intensity on the plate is 500 &mu;W/cm2 4. Remove the aluminum cover and lid and cover and expose the plate containing the TMP treated worms to the long wave UV radiation for 50 seconds.</li>
	<li>Cover and leave the worms in the dark for 5 hours at room temperature.</li>
	<li>Pick and transfer healthy looking J4 larvae to fresh seeded plates. Discard the original plate following your institution's hazardous chemical guidelines.</li>
	<li>Using a stereomicroscope, screen the F2 generation phenotypes for the mutation of interest.</li>
</ol>
Part 7: Representative Results
Some of the P. pacificus mutant phenotypes resulting from the mutagenesis (either by EMS or TMP/UV) are shown in Figure 1. Each mutant has a characteristic phenotype that is easily distinguishable from the wild type animal. From the worms that were treated with the mutagen, 30 J3/J4 larvae from the parental generation (P0) were placed in separate plates and allowed to lay eggs, which are the F1 generation. From the plates that showed healthy F1 progeny, a total of 1000 F1s were transferred on to individual plates and the F2 progeny was screened for phenotypes. Animals with Dumpy 5,6, Uncoordinated6 and Transformer7 phenotypes were found.
<img src="/files/ftp_upload/2293/2293fig1.jpg" alt="Figure 1" /> Figure 1. Pristionchus pacificus wild type and mutant animals. A. wt hermaphrodite B. wt male C. dpy mutant D. unc mutant E. tra mutant . Top two panels showing wild type hermaphrodite (left )and male (right) animals to compare with the different mutants in the remaining panels.

<ol>
	<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetics. 77 (1), 71-71 (1974).</li><li>Anderson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods Cell Biol. 48, 31-31 (1995).</li><li>Yandell, M. D., Edgar, L. G., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (4), 1381-1381 (1994).</li><li>Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WormBook. , 1-1 (2005).</li><li>Kenning, C., Kipping, I., Sommer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Genesis. 40 (3), 176-176 (2004).</li><li>Sommer, R. J., Carta, L. K., Kim, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamental and Applied Nematology. 19 (6), 511-511 (1996).</li><li>Pires-daSilva, A., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genes &#38; development. 18 (10), 1198-1198 (2004).</li><li>Wei, A., Yuan, A., Fawcett, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nucleic acids research. 30 (20), 110-110 (2002).</li><li>Jorgensen, E. M., Mango, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nat Rev Genet. 3 (5), 356-356 (2002).</li></ol>

No conflicts of interest declared.

Mutagenesis is a widely employed technique for genetic studies in various experimental model organisms. Mutagenesis experiments are often used to identify the function of a gene2. The mutagenesis procedures described here can be used not only for P. pacificus, but also for C. elegans and other related nematode species. Here we describe two methods, EMS and TMP/UV mutagenesis.
EMS is a chemical that induces point mutations or small deletions throughout the genome<s...

an introduction to worm lab: from culturing worms to mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Watch this Scientific Journal Video about An Introduction to Worm Lab: from Culturing Worms to Mutagenesis at JoVE.com

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

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34.2K Views.  University of Texas at Arlington. Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

Video: An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

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Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...