Name: an in vitro skin irritation test (sit) using the epiderm reconstructed human epidermal (rhe) model
Uploaded: 2009-07-13T21:00:00
Duration: 21 min 16 s
Description: Watch this Scientific Journal Video about An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model at JoVE.com

The authors would like to thank to ZEBET at the BfR (Germany), BASF SE (Germany), IIVS Inc. (USA), Zet-LSL (Austria) for participation in Multi-center International Validation Study of the Modified EpiDerm SIT (5).

I. Tissue conditioning - Day 0
 <ol>
 <li>Upon receipt of the EpiDerm EPI-200-SIT kit, check all the kit components for integrity (for kit details see Table of specific reagents and equipment). </li>
 <li>For every three EpiDerm tissues, prepare one sterile 6-well plate pre-filled with 0.9 ml of assay medium. </li>
 <li>Under sterile conditions, open the plastic bag containing the 24-well plate containing the EpiDerm tissues and remove the sterile gauze. </li>
 <li>Use sterile forceps to remove each insert containing the EpiDerm tissue and place the insert in the empty 24-well plate. During this step, remove any remaining shipping agarose that adheres to the outer sides of the insert by gentle blotting on sterile filter paper.</li>
 <li>Within the next 5 minutes, visually inspect the tissues. Do not use defective tissues or tissues which are completely covered with liquid. </li>
 <li>Using a sterile cotton tip swab, carefully dry the surface of the tissues. Try not to touch the tissue directly - capillary effects are sufficient to remove the moisture from the tissue surface. </li>
 <li>After drying the tissue surface, transfer three tissues to the top row of the 6-well plates pre-filled with 0.9 mL of assay medium. Release any air bubbles trapped underneath the inserts. </li>
 <li>Place the 6-well plates containing the inserts into the incubator for a 60 &plusmn; 5 min pre-incubation at 37 &plusmn; 1&deg; C, 5 &plusmn; 1% CO2, 95 % RH(Relative Humidity).</li>
 <li>At the end of the 60 &plusmn; 5 min pre-incubation period, transfer the inserts from the upper wells into the lower wells of the 6-well plate. Place the plates back into the incubator (37 &plusmn; 1&deg; C, 5 &plusmn; 1% CO2, 95 % RH) for an overnight pre-incubation (18 &plusmn; 3 hr).</li>
 <li>Place the rest of the assay medium into the refrigerator (5 &plusmn; 3 &deg;C). Following the overnight pre-incubation, the test chemicals can be applied to the EpiDerm tissues.
 Note: The pre-incubation procedure can be shortened in case of late tissue delivery (e.g. if tissues arrive on Wednesday instead of Tuesday). 
</li>
 </ol>
 II. Chemical exposure - Day 1
 <ol>
 <li>Prepare one 6-well plate per test chemical by filling the upper row only with 0.9 ml per well of assay medium.</li>
 <li>Approximately 5 minutes before the planned exposure to chemicals, remove the 6-well plates containing the pre-conditioned tissues from the incubator. </li>
 <li>Evaluate the surface of tissues and remove any moisture using sterile cotton tips.</li>
 <li>Label all 6-well plate lids with the test material codes or names.</li>
 <li>Dose the tissues with the test chemicals at 1 minute intervals to facilitate rinsing of the test chemicals after exposure. Keep the plates with dosed tissues in the sterile hood, until the last tissue is dosed. </li>
 <li>To test liquid chemicals, dispense 30 &mu;L of the solution directly atop the tissue and place the nylon mesh on the tissues surface. If necessary, gently position the mesh using the bulb headed glass Pasteur pipette. Do not press on the tissue surface.</li>
 <li>For testing semisolids, use a positive displacement pipette to dispense 30 &mu;L directly atop the tissue. If necessary spread the chemical with Pasteur pipette to cover the tissue surface as much as possible.</li>
 <li>For solid materials, shortly before application of the test substance, moisten the tissue surface with 25 &mu;L of sterile DPBS. This will improve contact of the tissue surface with the test chemical. Fill a 25 mg calibrated sharp application spoon (or any other appropriate tool) with approximately 25 mg of finely ground test material. Apply the solid test chemical to the tissue surface. If necessary, use a bulb headed Pasteur Pipette to empty the spoon completely. Gently shake the inserts to improve the spreading of the solid on the surface.</li>
 <li>For test substances with waxy consistency, try to form a flat "cookie like" piece about 8 mm in diameter and place it atop the tissue, which was previously wetted with sterile DPBS. To improve the contact between test substance and tissue, weigh down the "cookie" with a stainless steel or plastic aid.</li>
 <li>Apply DPBS as the negative control and 5% SDS as the positive control to the control tissues.</li>
 <li>After dosing the last tissue, transfer all the plates for 35 &plusmn; 1 minutes to the humidified incubator (37 &plusmn; 1&deg; C, 5 &plusmn; 1% CO2, 95 % RH).</li>
 <li>After the 35 &plusmn; 1 minute incubation at 37&deg;C remove all the plates from the incubator. Place the plates into the sterile hood and wait until a period of 60 minutes chemical exposure is completed for the first dosed tissue.</li>
 <li>Afterwards start the washing procedure using the one insert per minute time interval (to insure that the total exposure time for every insert is 60 minutes).</li>
 <li>Use a wash bottle to rinse the tissues with sterile DPBS. Fill the tissue insert 15 times by using a constant stream of DPBS and empty it (Note: for tissues with a nylon mesh, wash the tissue surface 5 times and remove the mesh carefully with the pointed, sharp forceps. Afterwards, continue with the remaining 10 washes).</li>
 <li>After the 15th rinse using the wash bottle, completely submerge the insert 3 times in 150 ml DPBS and shake to remove the remainder of the test material.</li>
 <li>Finally, rinse the tissue insert once from the inside and once from the outside with sterile DPBS. </li>
 <li>Remove any excess of DPBS from the tissue by gently shaking the insert, and blotting it on the sterile blotting paper. </li>
 <li>Transfer the blotted tissue inserts to the new 6-well plate previously pre-filled with assay medium. All of these steps (14-18) must be done within 1 min for each insert.</li>
 <li>After the last tissue is rinsed, carefully dry the surface of each tissue with a sterile cotton tipped swab. </li>
 <li>Incubate the tissues in the incubator for the next 24 &plusmn; 2 hours (37 &plusmn; 1&deg; C, 5 &plusmn; 1% CO2, 95 % RH).
 Note: Each assay technician should not test more than 6 test substances including the negative and positive controls in a single testing run (set), to be able follow the protocol as described below</li>
</ol>
III. Medium exchange - Day 2
 <ol>
 <li>After the 24 &plusmn; 2 hour post-incubation, transfer the inserts into the lower part of the 6-well plate, pre-filled with 0.9 ml of media. Media from upper rows can be collected for analysis of the additional endpoints (cytokines, chemokines etc.). Analysis of the secondary endpoints is optional.</li>
 <li>Continue with the post-incubation (37 &plusmn; 1&deg; C, 5 &plusmn; 1% CO2, 95 % RH) for another 18 &plusmn; 3 hours.</li>
 </ol>
IV. MTT Viability Assay - Day 3
 <ol>
 <li>Label two 24-well plates with the chemical names or codes. One plate will serve for tissue incubation with MTT and the other for the extraction step.</li>
 <li>Prepare the MTT solution by thawing the MTT concentrate (5 mg/ml) and diluting it with the MTT diluent. Final concentration of MTT solution is 1 mg/ml. </li>
 <li>Pipette 300 &mu;L of MTT solution into each well of the 24 well plate.</li>
 <li>Remove the 6-well plates from incubator, blot the bottom of the inserts on a blotting paper, and transfer them into the 24-well plate pre-filled with MTT.</li>
 <li>Place the 24-well plate in the incubator (37 &plusmn; 1&deg; C, 5 &plusmn; 1% CO2, 95 % RH) and incubate for 3 hours &plusmn; 5 min. Strictly adhere to the 3 hours &plusmn; 5 min incubation time to avoid deviation between different MTT readings. </li>
 <li>When the MTT incubation is complete, use a suction pump to gently aspirate the MTT medium from all the wells. </li>
 <li>Refill the wells with DPBS and aspirate again. Repeat this rinsing two more times and make sure that tissues are dry after the last aspiration. </li>
 <li>After the washes transfer the inserts to a new 24-well plate. Immerse the inserts by gently pipetting 2 mL extractant solution (Isopropanol) into each insert. The level will rise above the upper edges of the insert, thus completely submerging the tissues. </li>
 <li>Seal the 24-well plate (e.g. with Parafilm or using sealing bag) to inhibit extractant evaporation. Extract the MTT from the tissues for at least 2 hours at room temperature with gentle shaking on a plate shaker (~ 120 rpm). Overnight extraction without shaking can also be used. </li>
 <li>After the extraction period is complete, pierce the inserts with an injection needle or bulb beaded Pasteur pipette and allow the extract to run into the well from which the insert was taken.</li>
 <li>Discard the punctured insert and pipette the solution in the well up and down three times until it is homogenous. </li>
 <li>For each tissue, transfer two 200 &mu;L aliquots of the purple formazan solution into a 96-well flat bottom microtiter plate. Transfer the duplicates according to the fixed plate design given in the spreadsheet accompanying this video protocol. For blanks, use isopropanol. </li>
 <li>Read the optical density (OD) of the MTT extracts in a 96-well plate spectrophotometer using a wavelength of 570 nm (540-580) without a reference filter.</li>
 <li>Enter the results in the spreadsheet for automatic calculation of the results (Figure 1). </li>
 <li>A test chemical is labeled 'irritant' (R38 or GHS category 2) if the percent tissue viability determined by MTT assay is &lt;50% relative to the negative control. 
 Note: MTT is toxic, so be sure to wear protective gloves when handling. Protect MTT and its solutions from light, since MTT is light sensitive. Use MTT solution within a few hours after preparation because it degrades over time.</li></ol>
 V. Assay Quality Controls
<ul>
<li> EpiDerm EPI-200-SIT kit Acceptance Criteria: 
In vitro RHE models should provide consistent, high quality data over time, not just during the validation process (8). Recommended guidelines to ensure long-term assay reproducibility and performance include "full characterization of cells or tissues, sampling of each lot ... for performance, and regular use of controls and benchmark chemicals to provide assurance of consistency of assay performance" (9). In addition to assay acceptance criteria outlined below, each production lot of EpiDerm is quality control tested by the manufacturer against an additional benchmark chemical (1% Triton X-100). The exposure time needed to reduce tissue viability to 50% (ET-50) for Triton X-100 is determined. For EpiDerm, yearly average ET-50 values have ranged from 5.9 hrs to 7.5 hrs. The lot to lot CV for negative control tissues (i.e. the variability in baseline viability) has averaged under 7.5% for every year since 1996. The ET50 value of each production lot must fall within 2 standard deviations of the historical average Triton X-100 ET50 established in 1996 (i.e. ET-50 = 6.7 hours; Lower acceptance limit = 4.8h ; upper acceptance limit = 8.7h) in order to be released for use by the manufacturer.</li>
<li>EPI-200-SIT Assay Acceptance Criterion 1: Negative Control: 
The absolute OD of the negative control (NC) tissues (treated with sterile DPBS) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is &ge;1.0 and &le;2.5.</li>
<li>EPI-200-SIT Assay Acceptance Criterion 2: Positive Control 
A 5% SDS (in H2O) solution is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means that the PC has to be tested in each assay, but not more than one PC is required per testing day. Viability of positive control should be within 95% confidence interval of the historical data. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is 20%.</li>
<li>EPI-200-SIT Assay Acceptance Criterion 3: Standard Deviation (SD) 
The skin irritancy is predicted from the mean viability determined on 3 single tissues and therefore the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the SD calculated from individual % tissue viabilities of the 3 identically treated replicates is &lt;18%.</li>
</ul>
Representative Results
<img src="/files/ftp_upload/1366/1366fig1.jpg" alt="Figure 1" /> <img src="/files/ftp_upload/1366/1366fig1_2.jpg" alt="Figure 2" /> <img src="/files/ftp_upload/1366/1366fig1_3.jpg" alt="Figure 3" /> Figure 1. Results obtained for 6 test articles, NC and PC control - Part of the automated spreadsheet for calculation of results. Test chemicals which reduced tissue viability below 50% referenced to NC are classified as irritants. The test is optimized to provide results that are sufficiently far from the classification cut-off (50% viability) thus increasing the robustness of the assay.
<img src="/files/ftp_upload/1366/materialstable.jpg" alt="Materials List" />

<ol>
	<li>Kand&#225;rov&#225;, H., Liebsch, M., Genschow, E., Gerner, I. n. g. r. i. d., Traue, D., Slawik, B., Spielmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+EpiDerm+Test+Protocol+for+the+upcoming+ECVAM+Validation+Study+on+In+Vitro+Skin+Irritation+Tests.">Optimization of the EpiDerm Test Protocol for the upcoming ECVAM Validation Study on In Vitro Skin Irritation Tests.</a> ALTEX. 21, 107-114 (2004).</li><li>Kand&#225;rov&#225;, H., Liebsch, M., Gerner, I., Schmidt, E., Genschow, E., Traue, D., Spielmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EpiDerm+Skin+Irritation+Test+Protocol+-+Assessment+of+the+performance+of+the+optimized+test.">EpiDerm Skin Irritation Test Protocol - Assessment of the performance of the optimized test.</a> ATLA. 33, 351-367 (2005).</li><li>Kand&#225;rov&#225;, H., Hayden, P., Klausner, M., Kubilus, J., Kearney, P., Sheasgreen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Skin+Irritation+Test:+Improving+the+Sensitivity+of+the+EpiDerm+Skin+Irritation+Test+Protocol.Accepted+for+publication.">In Vitro Skin Irritation Test: Improving the Sensitivity of the EpiDerm Skin Irritation Test Protocol.Accepted for publication.</a> ATLA. , (2009).</li><li>Spielmann, H., Hoffmann, S., Liebsch, M., Botham, P., Fentem, J., Eskes, C., Roguet, R., Cotovi, J., Cole, T., Worth, A., Heylings, J., Jones, P., Robles, C., Kand&#225;rov&#225;, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ECVAM+International+Validation+Study+on+In+Vitro+Tests+for+Acute+Skin+Irritation:+Report+on+the+Validity+of+the+EPISKIN+and+EpiDerm+Assays+and+on+the+Skin+Integrity+Function+Test.">The ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Report on the Validity of the EPISKIN and EpiDerm Assays and on the Skin Integrity Function Test.</a> ATLA. 35, 559-601 (2007).</li><li>Liebsch, M., Gamer, A., Curren, R., Frank, J., Genschow, E., Tharmann, J., Remmele, M., Bauer, B., Raabe, H., Barnes, N., Hilberer, A., Wilt, N., Lornejad-Sch&#228;fer, M. R., Sch&#228;fer, C., Spiller, E., Hayden, P., Kand&#225;rov&#225;, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follow-up+validation+of+the+modified+EpiDerm+Skin+Irritation+Test+(SIT):+results+of+a+multicentre+study+of+twenty+reference+test+substances.">Follow-up validation of the modified EpiDerm Skin Irritation Test (SIT): results of a multicentre study of twenty reference test substances.</a> , (2008).</li><li>United Nations. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Globally Harmonized System of Classification and Labeling of Chemicals (GHS). , (2007).</li><li>ESAC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Statement on the Scientific Validity of In-Vitro Tests for Skin Irritation Testing. , (2008).</li><li>Gupta, K., Rispin, A., Stitzel, K., Coecke, S., Harbell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ensuring+quality+of+in+vitro+alternative+test+methods:+Issues+and+answers.">Ensuring quality of in vitro alternative test methods: Issues and answers.</a> Regul Toxicol Pharmacol. 43, 219-224 (2005).</li><li>Rispin, A., Stitzel, K., Harbell, J., Klausner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ensuring+quality+of+in+vitro+alternative+test+methods:+Current+practice.">Ensuring quality of in vitro alternative test methods: Current practice.</a> Regul Toxicol Pharmacol. 45, 97-103 (2006).</li></ol>

The authors are employees of MatTek corporation which produce reagents and tools used in this article.

In this video, we have demonstrated the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients. When performing this method, it is important to work in aseptic conditions and to strictly follow the validated protocol, since deviation from the protocol may cause different outcomes. Some modifications of the assay are possible, however, changes to the protocol should be discussed directly with the authors.
The only limitation of this method is a possible interference of the test substance with the MTT endpoint. A colored test substance or one that directly reduces MTT (and thereby mimics dehydrogenase activity of the cellular mitochondria) may interfere with the MTT endpoint. However, these test substance are a problem only if at the time of the MTT test (i.e. 42 hours after test substance exposure) sufficient amounts of the test substance are still present on (or in) the tissues. In case of this unlikely event, the (true) metabolic MTT reduction and the contribution by a colored test material or (false) direct MTT reduction by the test material can be quantified by a procedure described in detail in the assay Standard Operation Procedure provided by MatTek Corp.

an in vitro skin irritation test (sit) using the epiderm reconstructed human epidermal (rhe) model

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients. The EpiDerm SIT utilizes the 3D in vitro reconstructed human epidermal (RHE) model EpiDerm. The procedure described in this protocol allows for discrimination between irritants of GHS category 2 and non-irritants (6). The test is performed over the course of a 4 day time period, consisting of pre-incubation, 60 minute exposure, 42 hour post-incubation and MTT viability assay. After tissue receipt and overnight pre-incubation (Day 0), tissues are topically exposed to the test chemicals (Day 1), which can be liquid, semi-solids, solid or wax. Three tissues are used for each test chemical, as well as for the positive control (5% aq. SDS solution), and a negative control (DPBS). Chemical exposure lasts for 60 minutes, 35 min of which the tissues are kept in an incubator at 37&deg;C. The test substances are then removed from the tissue surface by an extensive washing procedure. The tissue inserts are blotted and transferred to fresh medium. After a 24 hr incubation period (Day 2), the medium is exchanged. The medium can be saved for further analysis of cytokines or other endpoints of interest. After the medium exchange, tissues are incubated for an additional 18 hours. At the end of the entire 42h post-incubation (day 3), the tissues are transferred into yellow MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, is extracted for 2 hours using isopropanol. The optical density of the extracted formazan is determined using a spectrophotometer. A chemical is classified as an irritant if the tissue viability relative to the negative control treated tissues is reduced below 50%. This procedure can be used as full replacement of the in vivo rabbit skin irritation test for hazard identification and labeling of chemicals in line with EU regulations (7).

Watch this Scientific Journal Video about An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model at JoVE.com

An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model

In this video, we demonstrate the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients.

An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including ...

an-vitro-skin-irritation-test-sit-using-epiderm-reconstructed-human

Research

JoVE Journal

Biology

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...