Name: Quantification of Colonic Stem Cell Mutations (Video) | JoVE Journal
Uploaded: 2015-09-25T15:00:00.000+00:00
Duration: 7 min 53 s
Description: Learn to quantify somatic mutations in colonic stem cells using G6PD staining and light microscopy in treated mice. This method enables precise measurement of genotoxic effects on stem cell populations.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors have no acknowledgements.

The experimental procedures and ethical treatment of animals was approved by the University of Pittsburgh IACUC (protocol #1104674).
1. Preparation of G6PD Staining Mixture
NOTE: Ensure that the final concentration of each reagents is as follows; 5 mM glucose-6-phosphate (G6P); 2 mM NADP, 5 mM MgCl2, 0.35 mM 1-methoxy-5-methylphenazinium methyl sulfate (MMPMS) and 0.8 mM NBT.1,8,9 For each reagent the initial concentration was derived for a 40 ml final volume. Solutions were freshly prepared and used each day.
<ol>
	<li>Add 2 ml of 200 mM pH 7.4 phosphate buffer (PB) to 35 ml of OCT medium in a 50 ml tube and shake vigorously. Confirm with pH paper that the solution is ~pH 7.4. If not discard the solution.</li>
	<li>Add 61 mg G6P dissolved in 0.94 ml of 200 mM PB, 61 mg NADP dissolved in 0.94 ml of 200 mM PB, and 41 mg MgCl2 dissolved in 0.96 ml of 100 mM PB. Reconfirm that the pH is ~7.4 with pH paper. If not ~7.4, discard the solution.</li>
	<li>Add 5 mg of MMPMS dissolved in 0.25 ml of 200 mM PB (pH 7.4). Vigorously shake the resulting reaction mixture to generate a clear dark red homogenate. If the solution is turbid, the mixture should be discarded. The tube containing the reaction mixture is wrapped in foil to minimize exposure to light. Store the reaction mixture at RT while the final component, NBT, is prepared.</li>
	<li>Prepare separately a 5 mM NBT solution by adding 0.4 ml anhydrous dimethyl formamide (DMF) and 0.4 ml anhydrous ethanol to 164 mg of NBT in a 2 ml tube with an O-ring lid. Close the lid loosely (not tightly sealed) and bring the solution to a vigorous boil in an oil bath until the NBT is in solution.8</li>
	<li>Allow the solubilized NBT to cool to RT and then add all of the solution to the OCT reaction mixture and shake vigorously to obtain a clear solution. Observe the solution color change from initial orange-red to a dark-red or purple color over time. This is normal.</li>
	<li>Place the final G6PD staining mixture in a 37&#160;&#176;C warm room for 1 hr before use.</li>
</ol>
2. Animal Treatment and Tissue Collection
<ol>
	<li>Treat 6-week-old C57Bl/6 male mice with DNA damaging agent, e.g., 10 mg/kg azoxymethane (AOM) in a volume of 200 &#181;l PBS by i.p. injection. Treat control mice with 200 &#181;l of PBS by i.p. injection.</li>
	<li>At 90 days, euthanize mice by CO2 asphyxiation followed by cervical dislocation as approved by IACUC protocol.</li>
	<li>Surgically open the abdominal cavity from just above the anus to the base of the sternum. Move the small intestine out of the body cavity but do not excise it. Excise the lower intestine just below the cecum to the rectum.10</li>
	<li>Carefully slice along one side of the colon using a micro-dissection scissor and remove feces from the opened colon by washing with clean sterile PBS. Swiss roll the excised colon with the luminal side facing outward, and place in a tissue mold.10</li>
	<li>Completely immerse the colon in OCT medium and place mold in a Dewar containing liquid N2. Hold the mold so that the plastic base is just in contact with the liquid N2 until the OCT medium is frozen solid.</li>
	<li>Store the frozen tissue block at -80 &#160;until cutting frozen sections. Do not use any fixative, which would result in the inactivation of the G6PD enzyme.</li>
</ol>
3. Preparation of Frozen Sections
<ol>
	<li>Cut frozen tissue sections at a thickness of 7 &#181;m using a cryostat at -25 &#176;C. Cut 4 consecutive 7 &#181;m sections and place two of them on the same slide (Figure 1). Prepare 2 slides, or four consecutive 7 &#181;m sections to represent one level of the colon (Figure 1).</li>
	<li>Repeat for the next level with a minimum distance of &#62;50 &#181;m from the last section of the previous level. This distance ensured that the crypts assessed for each level did not duplicate one another.</li>
	<li>Frequently wipe the cryostat blade with 100% ethanol to remove the OCT residue that accumulates during cutting. Before sectioning is resumed, the dry blade is wiped with an antistatic dryer sheet to dissipate accumulated static charge. Store slides with the frozen sections at -80 oC.</li>
</ol>
4. Staining of Frozen Section Tissue
<ol>
	<li>Perform the enzyme histochemistry reaction in a 37 &#176;C&#160;warm room with high humidity. 
	NOTE: Attempting the enzymatic reaction in an incubation oven or on a slide warmer led to poor and inconsistent G6PD staining.</li>
	<li>Build wells for tissue staining using two 1.5 cm x 0.15 cm steel washers (interior diameter x height) that are bonded together using silicone grease (Figure 1). Cut the parafilm to fit the base of the well with the center cut out and the well placed over the tissue section on the slide. 
	NOTE: The parafilm on the bottom of the washer creates a seal between the slide and the washer that maintains the viscous G6PD staining mixture on the tissue section. This construct gives a well with a 0.4 - 0.5 ml volume so that each tissue section in the study receives the same volume of the G6PD reaction staining mixture.</li>
	<li>Incubate frozen tissue slides at 37 oC in a warm room for 10 min. At 37 &#176;C, add the G6PD staining reaction mixture to each tissue section well (2 sections per slide) for 45-50 min. Remove slides from warm room, and carefully lift the stainless steel wells off the slides.</li>
	<li>Set slides on their long edge to allow the reaction medium to drain. Place slides in 100 mM PB (pH 7.4) for 30-60 min to remove the G6PD staining reaction mixture from the tissue without disturbing the tissue staining. Submerge slides in distilled water for 5-10 min to remove PB salts</li>
	<li>Seal the tissue by adding fluoro-gel from a dropper onto the tissue and waiting ~ 30 min for it to dry. Avoid bubbles that can confound the identification of mutated crypts. 
	NOTE: If bubbles form, the procedure can be repeated after removal of the fluoro-gel by soaking in distilled water. Do not use cover slips as they tend to trap air bubbles.</li>
</ol>
5. Determination of G6PD Mutant Crypts and Mutation Frequency
<ol>
	<li>Analyze slides for G6PD mutant crypts under a light microscope. 
	NOTE: The criteria used to identify a fully mutant crypt were: (i) the entire crypt was devoid of blue color; (ii) the outer structure of the crypt was intact, (iii) the mutant crypt was observed on adjacent 7 &#181;m sections and, (iv) two observers had to identify the same crypt as a mutant. Mutants observed on adjacent 7 &#181;m sections are counted as one mutant.</li>
	<li>Image each G6PD mutant crypt at 40x magnification and catalogue the images using a 5.0 megapixel camera with imaging software.</li>
	<li>Quantify the total number of crypts examined in a mouse by selecting a representative section for each mouse from the level with the smallest surface area and image it at 10x magnification (Figure 1C). 
	NOTE: A level represents four consecutive 7 &#181;m sections (Figure 1D), which due to their proximity often show mutated crypts on more than one section. The levels are separated by &#62;50 &#181;m to visualize crypts from different areas of the colon.</li>
	<li>Open each image file and visually count the number of crypts on a level by analyzing the files (shown as boxes in Figure 1C). Images files will vary in the number of crypts observed from as low as 20 to &#62;300 crypts in one file.</li>
	<li>Multiply the total number of crypts from the selected level (Figure 1C) for each mouse by the number of levels screened for G6PD mutant crypts for each colon. For example, a total count of 1,250 crypts per selected level multiplied by 8 levels screened for mutant crypts equals a total count of 10,000 crypts. Evaluate a minimum of 10,000 crypts for each colon for statistical analysis.</li>
	<li>Combine the number of mutants observed in all of the animals by the total number of crypts screened to calculate the MF for each treatment. The background stem cell MF level in untreated mice is &#60; 0.1x10-4 (Table 1), which is close to that previously reported in C57Bl/6 mice.11</li>
</ol>

<ol><li>Griffiths, D. F., Davies, S. J., Williams, S., Williams, G. T., Williams, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Demonstration+of+somatic+mutation+and+colonic+crypt+clonality+by+X-linked+enzyme+histochemistry%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Demonstration of somatic mutation and colonic crypt clonality by X-linked enzyme histochemistry.</a> Nature. 333 (6172), 461-463 (1988).</li>
<li>Griffiths, D. F., Sacco, P., Williams, G. T., Williams, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+clonal+origin+of+experimental+large+bowel+tumours%5BTitle%5D)+AND+%22Br.+J.+Cancer%22%5BJournal%5D)">The clonal origin of experimental large bowel tumours.</a> Br. J. Cancer. 59 (3), 385-387 (1989).</li>
<li>Kuraguchi, M., Thomas, G. A., Williams, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Somatic+mutation+of+the+glucose-6-phosphate+dehydrogenase+%28g6pd%29+gene+in+colonic+stem+cells+and+crypt+restricted+loss+of+G6PD+activity%5BTitle%5D)+AND+%22Mutat.+Res%22%5BJournal%5D)">Somatic mutation of the glucose-6-phosphate dehydrogenase (g6pd) gene in colonic stem cells and crypt restricted loss of G6PD activity.</a> Mutat. Res. 379 (1), (1997).</li>
<li>Van Noorden, C. J., Vogels, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polyvinyl+alcohol+and+other+tissue+protectants+in+enzyme+histochemistry%3A+a+consumer%E2%80%99s+guide%5BTitle%5D)+AND+%22Histochem.+J%22%5BJournal%5D)">Polyvinyl alcohol and other tissue protectants in enzyme histochemistry: a consumer’s guide.</a> Histochem. J. 21 (7), 373-379 (1989).</li>
<li>Giardina Rosenberg, D. W., C,, Tanaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+for+the+study+of+colon+carcinogenesis%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">Mouse models for the study of colon carcinogenesis.</a> Carcinogenesis. 30 (2), 183-196 (2009).</li>
<li>Tanaka, T., Kohno, H., Suzuki, R., Yamada, Y., Sugie, S., Mori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+inflammation-related+mouse+colon+carcinogenesis+model+induced+by+azoxymethane+and+dextran+sodium+sulfate%5BTitle%5D)+AND+%22Cancer+Sci%22%5BJournal%5D)">A novel inflammation-related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate.</a> Cancer Sci. 94 (11), 965-973 (2003).</li>
<li>Suzuki, R., Kohno, H., Sugie, S., Tanaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dose-dependent+promoting+effect+of+dextran+sodium+sulfate+on+mouse+colon+carcinogenesis+initiated+with+azoxymethane%5BTitle%5D)+AND+%22Histol.+Histopathol%22%5BJournal%5D)">Dose-dependent promoting effect of dextran sodium sulfate on mouse colon carcinogenesis initiated with azoxymethane.</a> Histol. Histopathol. 20 (2), 483-492 (2005).</li>
<li>Frederiks, W. M., Vreeling-Sindalarova, H., Van Noorden, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+peroxisomes+causes+oxygen+insensitivity+of+the+histochemical+assay+of+glucose-6-phosphate+dehydrogenase+activity+to+detect+cancer+cells%5BTitle%5D)+AND+%22J.+Histochem.+Cytochem%22%5BJournal%5D)">Loss of peroxisomes causes oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase activity to detect cancer cells.</a> J. Histochem. Cytochem. 55 (2), 175-181 (2007).</li>
<li>Hisada, R., Yagi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((1-Methoxy-5-methylphenazinium+methyl+sulfate%5BTitle%5D)+AND+%22Biochem%22%5BJournal%5D)">1-Methoxy-5-methylphenazinium methyl sulfate.</a> Biochem. 82 (5), 1469-1473 (1977).</li>
<li>Whittem, C. G., Williams, A. D., Williams, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Murine+Colitis+modeling+using+Dextran+Sulfate+Sodium+%28DSS%29%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Murine Colitis modeling using Dextran Sulfate Sodium (DSS).</a> J Vis Exp. (35), (2010).</li>
<li>Kuraguchi, M., Cook, H., Williams, E. D., Thomas GA, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differences+in+susceptibility+to+colonic+stem+cell+somatic+mutation+in+three+strains+of+mice%5BTitle%5D)+AND+%22J.+Pathol%22%5BJournal%5D)">Differences in susceptibility to colonic stem cell somatic mutation in three strains of mice.</a> J. Pathol. 193 (4), 517-521 (2001).</li>
<li>Whetstone, R. D., Gold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T-Cells+Enhance+Stem+Cell+Mutagenesis+in+the+Mouse+Colon%5BTitle%5D)+AND+%22Mutat.+Res%22%5BJournal%5D)">T-Cells Enhance Stem Cell Mutagenesis in the Mouse Colon.</a> Mutat. Res. 744, 1-5 (2015).</li>
<li>Van Noorden, C. J., Vogel, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histochemistry+and+cytochemistry+of+glucose-6-phosphate+dehydrogenase%5BTitle%5D)+AND+%22Prog.+Histochem.+Cytochem%22%5BJournal%5D)">Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase.</a> Prog. Histochem. Cytochem. 15 (4), 1-85 (1985).</li>
<li>Van Noorden, C. J., Vogels, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+sensitive+cytochemical+staining+method+for+glucose-6-phosphate+dehydrogenase+activity+in+individual+erythrocytes.+II.+Further+improvements+of+the+staining+procedure+and+some+observations+with+glucose-6-phosphate+dehydrogenase+deficiency%5BTitle%5D)+AND+%22Br.+J.+Haematol%22%5BJournal%5D)">A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes. II. Further improvements of the staining procedure and some observations with glucose-6-phosphate dehydrogenase deficiency.</a> Br. J. Haematol. 60 (1), 57-63 (1985).</li>
<li>Cook, H. A., Williams, D., Thomas, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crypt-restricted+metallothionein+immunopositivity+in+murine+colon%3A+validation+of+a+model+for+studies+of+somatic+stem+cell+mutation%5BTitle%5D)+AND+%22J.+Pathol%22%5BJournal%5D)">Crypt-restricted metallothionein immunopositivity in murine colon: validation of a model for studies of somatic stem cell mutation.</a> J. Pathol. 191 (3), 306-312 (2000).</li>
</ol>

The authors have nothing to disclose.

Often the genotoxic effect of a compound is determined by its ability to modify DNA. This is normally done by isolating the tissue and measuring the global level of DNA adducts. For AOM, this would involve quantifying O6-methylguanine adducts in the colon. Using this approach information on damage within specific cell types, such as in the stem cell niche, is lost. In addition, a DNA adduct is not the same as a mutation since only a small subset of adducts are eventually converted into fixed mutations.12<...

Colon cancer is thought to involve exposure to environmental agents and dietary components that can damage DNA and produce activating somatic mutations in oncogenes (e.g., &#223;-catenin) or inactivating mutations in tumor suppressor genes (e.g., APC). It is assumed that these critical mutations occur in colonic stem cells. Because of the unique crypt architecture of the colonic epithelium, it is possible to measure stem cell mutations in the colon after exposing animals to chemicals associated with colon carcinogenesis. Several X-linked enzymes can serve as indicators for mutations that occur in stem cells, as the mutations will be present in all cells within a crypt.
Previously, a procedure was published demonstrating that chemicals that induce colon tumors in mice also generated somatic stem cell mutations in the colon via analysis of mutations in the X-linked glucose-6-phosphate dehydrogenase (G6PD) gene.1-3 The method quantifies the incidence of random somatic mutations in colonic stem cells without any selection pressure. The procedure involves the production of unfixed frozen colon sections from treated and control mice and the identification of crypts devoid of cells that produce functional G6PD activity. These mutated crypts, which appear white, indicate that the mutation occurred in a stem cell that gave rise to progeny also harboring a mutated G6PD gene. In the enzymatic assay, G6PD deficient mutant crypts cannot oxidize glucose-6-phosphate, which is required for the reduction of nitro blue tetrazolium (NBT). When the G6PD enzyme is functional, the NBT in the staining mixture is reduced to insoluble formazan and precipitates, accumulating at the location of the enzyme and &#8220;staining&#8221; cells blue. Cells that are G6PD mutants remain whitish in contrast to blue stained wild type crypts. This method measures mutations that have a &#8220;null&#8221; phenotype. Because enzymes, such as G6PD, can diffuse out of the cells on an unfixed tissue section if the sections are placed in an aqueous solution, it is necessary to stabilize the enzyme in the tissue sections to be analyzed.4 The stabilization of the enzyme in the tissue must not interfere with diffusion of small reagent molecules necessary for the G6PD enzymatic reaction.
We have made a number of significant changes to the original procedure. The medium for the critical enzymatic staining has been changed from polyvinyl alcohol to Optimal Cutting Temperature Compound (OCT), which facilitates pH control and solubilization of the components used in the assay. The staining is performed using 0.4 &#8211; 0.5 ml wells made of steel washers so that each tissue section received the same volume of the G6PD reaction mixture. A procedure was developed to estimate the number of crypts in an imaged section that provides a large sampling of the colonic tissue without having to manually count &#62;105 crypts for each colon. The analysis of adjacent 7 &#181;m tissue sections affords visualization of a mutant crypt on more than one slide, which reduces potential staining artifacts. These changes make the procedure more efficient and reproducible.
Using this revised protocol, we have quantified the mutation frequency in colonic stem cells after exposure of C57Bl/6 mice to azoxymethane, a well-known colon carcinogen in rodents.5-7

<table><tbody><tr><td>reagents</td><td></td><td></td><td></td></tr><tr><td>nitroblue tetrazolium</td><td></td><td></td><td></td></tr><tr><td>NADP</td><td></td><td></td><td></td></tr><tr><td>glucose-6-phosphate</td><td></td><td></td><td></td></tr><tr><td>1-methoxy-5-methylphenazinium methyl sulfate</td><td></td><td></td><td></td></tr><tr><td>dimethyl formamide</td><td></td><td></td><td></td></tr><tr><td>phosphate buffer (pH 7.4</td><td></td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature Compound (OCT)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>light microscope equipped with 5 megapixel camera</td><td></td><td></td><td></td></tr><tr><td>cryostat</td><td></td><td></td><td></td></tr><tr><td>warm room&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

quantification of  colonic stem cell mutations

Quantification of  Colonic Stem Cell Mutations

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce mutations that potentially lead to cancer. The use of an enzymatic assay to quantify stem cell mutations in the X-linked glucose-6-phosphate dehydrogenase gene has been previously reported.1 This method requires the preparation of frozen sections and incubation of the sectioned tissue with a reaction mixture that yields a blue color if the cells produce functional glucose-6-phosphate dehydrogenase (G6PD) enzyme. If not, the cells appear whitish. We have modified the reaction mixture using Optimal Cutting Temperature Compound (OCT) medium in place of polyvinyl alcohol. This facilitates pH measurement, increases solubilization of the G6PD staining components and restricts diffusion of the G6PD enzyme. To demonstrate that a mutation occurred in a stem cell, the entire crypt must lack G6PD enzymatic activity. Only if a stem cell harbors a phenotypic G6PD mutation will all of the progeny in the crypt lack G6PD enzymatic activity. To identify crypts with a stem cell mutation, four consecutive adjacent frozen sections (a level) were cut at 7 &#181;m thicknesses. This approach of making adjacent cuts provides conformation that a crypt was fully mutated since the same mutated crypt will be observed in adjacent sections. Slides with tissue samples that were more than 50 &#181;m apart were prepared to assess a total of &#62;104 crypts per mouse. The mutation frequency is the number of observed mutated (white) crypts &#247; by the number of wild type (blue) crypts in a treatment group.

The ability to measure colonic stem cell mutations in animals provides a unique way to correlate mutations to cancer induction. Normally, it is assumed that the critical step in carcinogenesis involves activating mutations in oncogenes and/or inactivating mutations in tumor suppressor genes. We injected C57Bl/6 mice with 200 µl of PBS or 10 mg/kg AOM in 200 µl of PBS. AOM is a known colon carcinogen.5-7 At 90 days the colons were analyzed for stem cell mutations. This time is required to allow stem cells to fu...

We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce ...

The overall goal of this procedure is to quantify phenotypic somatic mutations in the glucose six phosphate dehydrogenase or G six PD gene in colonic stem cells. After treatment of animals with putative genotoxic agents, this is accomplished by first treating animals with the agent waiting an appropriate amount of time to allow the progeny of a stem cell carrying a G six PD mutation to fully populate a crypt, sacrificing the animal, removing the colon, and forming it into a Swiss roll and freezing the tissue in liquid nitrogen. The second step is to prepare seven micron thick frozen sections on slides from the colon using a cryostat.The tissue is warmed to 37 degrees Celsius and then wells per tissue staining are constructed around the tissue sections. The final step is to incubate the tissue sections with the G six PD staining mixture and then analyze the sections under a light microscope cells with G six PD activity stain blue, while those with phenotypic mutations appear whitish. Ultimately the quantification of mutated crips is used to determine the mutation frequency.This technique measures glucose six phosphate dehydrogenase somatic mutations in stem cells, and this is the critical cell population in colon carcinogenesis. By identifying somatic stem cell mutations, it is possible to correlate mutation frequency to colon cancer incidents in treated animals. Though this method can provide insights into stem cell mutations in the colon, it can also be applied to other model organisms or organ systems.If a stem cell niche can be identified, Generally individuals new to this method will struggle with the preparation of the MBT solution.Solubilizing. The MBT solution requires careful attention to detailed instructions. To begin preparing the histological staining solution at two milliliters of pH 7.4 phosphate buffer to 35 milliliters of optimal cutting temperature, or OCT medium in a 50 milliliter tube and shake vigorously to combine using pH paper.Confirmed that the solution is approximately pH 7.4. Next at glucose six phosphate and a DP and magnesium chloride solution dissolved in phosphate buffer as indicated in the text protocol and again, confirmed that the pH of the solution remains approximately 7.4. ADD M-M-P-M-S solution and mix vigorously.This should result in a clear, dark red homogenate wrap the solution in foil to minimize exposure to light. If the solution becomes turbid, it should be discarded while an oil bath is heating. Separately, prepare a five millimolar nitro blue to teraz oleum or MBT solution by mixing 0.4 milliliters of dimethylformamide 0.4 milliliters of anhydrous ethanol and MBT in a two milliliter tube with an O-ring lid loosely seal the tube and bring it to a vigorous boil in the oil bath until the MBT is dissolved.Next, allow the MBT solution to cool to room temperature and then add it to the OCT reaction mixture. The solution may change color from the initial orange red to a dark red or purple over time. After treating animals and preparing frozen sections as indicated in the text protocol, build wells for tissue staining using two steel washers bonded together with silicone grease fit parfum to the base of the washer and cut out the center where the solution will be placed replaced.Pre-war the frozen tissue slides in a humid 37 degree Celsius room for 10 minutes. Next, place the wells on the slide to surround the tissue sections at approximately half a milliliter of the previously prepared G six PD staining mixture to each tissue section well and incubate for 45 to 50 minutes. Remove the slides from the warm room and carefully remove the stainless steel wells from the slides.Place the slides on the long edge and drain the reaction mix mixture, followed by a 30 to 60 minute wash and 100 millimolar phosphate buffer. To remove the final traces of the G six PD staining solution, rinse the slides and deionized water for five to 10 minutes to remove residual phosphate buffer. Finally, seal the tissue by adding fluoro gel dropwise to the slide and dry for 30 minutes to acquire images for later analysis.Place the slide on a light microscope and adjust to four DX magnification. Then take pictures of the stained tissue section with a 5.0 megapixel camera to estimate the total number of crypts examined. For each mouse, select a section from the level with the smallest surface area and image it at 10 x magnification.Count the number of mutant crips. According to the following criteria, the entire crypt is devoid of blue color. The outer structure of the crypt is intact.The mutant is observed on adjacent seven micron sections and two observers Identify the same crypt as mutant. Visually count the number of crypts in this section and multiply by the number of levels screened for each colon. Examples of mutant Crips observed in mice treated with the DNA damaging agent, azoxymethane can be seen here on the left you see a vertical or top-down view of a mutant crypt.While on the right is a horizontal or side view, for a crypt to be declared mutant, all of the cells within the crypt must remain. White crypts with a small amount of white are due to mucus production from goblet cells. Mutation frequency is calculated as the number of mutants observed divided by the total number of crypts screened no mutant.Crips were observed in colons from control mice representing a mutation frequency less than one times 10 to the minus fifth while azoxymethane treated mice showed a stem cell mutation frequency of 4.44 times 10 to the minus four Following this procedure. Other methods, like the genetic analysis of the type of the glucose x phosphate dehydrogenase mutations can be performed in order to answer additional questions like how the chemical agent miscodes for a mutation. After watching this video, you should have a good understanding of how to measure somatic stem cell mutations in mice treated with potential mutagenic and carcinogenic compounds.

quantification-of-colonic-stem-cell-mutations

Research

JoVE Journal

Developmental Biology

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray).  Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model. The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants spontaneously arisen or after exposure to genotoxins.

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Presented here is a protocol to discover the overexpressed driver genes maintaining the established cancer stem-like cells derived from colorectal HT29 cells. RNAseq with available bioinformatics was performed to investigate and screen gene expression networks for elucidating a potential mechanism involved in the survival of targeted tumor cells.

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

This protocol describes a simple and cost-effective method to investigate and quantify cell death in human colonic organoids in response to cytotoxic perturbagens such as cytokines. The approach employs a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software to quantify single-organoid responses to cytotoxic stimuli.

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

Learn to quantify somatic mutations in colonic stem cells using G6PD staining and light microscopy in treated mice. This method enables precise measurement of genotoxic effects on stem cell populations.

Quantification of Colonic Stem Cell Mutations

Explore More Videos