This work was supported in part by a DoD Breakthrough Award to AMW (BCRP W81XWH-14-1-0425). KCR was partially supported by intramural fellowships: the Pease Cancer Pre-Doctoral Fellowship and the Mary Galvin Burden Pre-Doctoral Fellowship in Biomedical Sciences and University of California, Riverside, intramural awards: the Graduate Council Fellowship Committee Dissertation Research Grant and the Graduate Division Dissertation Year Program Award. The authors thank Gillian M. Wright and Alyssa M. Kumari for assistance in early troubleshooting of this method.

All animal handling and procedures were approved by the University of California, Riverside institutional animal care and use committee and were in accordance with guidelines from the American Association for Laboratory Animal Care, the United States Department of Agriculture, and the National Institutes of Health. The described method utilized C57BL/6 adult, female mice. All animals were euthanized by decapitation prior to tissue harvesting.
NOTE: An overview of the protocol, which uses a blue dye to assist in efficient dissection and uncoiling of the oviduct, is shown in the first figure (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63168/63168fig01.jpg" /> 
Figure 1: Overview of the microdissection method. (A) Left panel shows the intact structure from which most of the ovarian fat pad and remnants of connective tissue surrounding the oviduct have been removed. After this, the addition of toluidine blue helps distinguish the coils of the oviduct (middle panel). Right panel shows structures after the removal of the ovary. (B) An uncoiled oviduct with internal turns for determining where each segment begins and ends. (C) Cartoon of the segmentation used (not drawn to scale). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63168/63168fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Preparation of the dissection surface
<ol>
	<li>Affix a piece of dental wax to a Petri dish with adhesive and let it dry (Figure 2A). Sanitize the dish with 70% ethanol. The surface is ready for dissection.</li>
</ol>
2. Macrodissection of the ovary, oviduct, and uterus
<ol>
	<li>Immobilize the Petri dish containing the dental wax on a cold platform of choice (e.g., an ice pack kept at -20 &#176;C prior to use) under a dissection microscope when ready for dissection (Figure 2A).</li>
	<li>Disinfect the ventral surface of the animal with 70% ethanol. Open the abdominal and pelvic cavity with sterile dissection scissors and move the gastrointestinal tract to one side to locate the dorsal bihornal uterus. Follow each uterine horn rostrally to locate each oviduct and ovary enveloped in the uterine fat pad, found just below the kidney (Figure 2B,C).</li>
	<li>Excise both lateral oviducts with the ovaries and a portion of the distal uterine horn still attached using dissection scissors. Cut each lateral uterine horn with approximately 1-2 cm of the horn still attached to the coiled oviduct and ovary (Figure 2D). Submerge the tissue immediately in 2 mL of cold dissection medium (see Table of materials).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63168/63168fig02.jpg" /> 
Figure 2: Macrodissection of the ovary and the dissection setup. The oviduct is located dorsally in the pelvic cavity at the base of the kidney within the ovarian fat pad (B). Locate the uterine bifurcation in the pelvic cavity (C) and follow the lateral uterine horn to locate the uterus-oviduct-ovary continuum (B). Remove these structures by cutting through one uterine horn and coarse dissection. Place onto the dissecting platform (A) and affix the uterine portion to dental wax with a needle (A and D). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63168/63168fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NOTE: Leave enough uterine horn intact and attached to the oviduct to allow affixation to the dissection platform (Figure 2D).
<ol start="4">
	<li>Affix the uterine tissue to the dental wax with a sterile 25 G needle to secure tissue for dissection (Figure 2A,D, and Figure 3A). Working under the dissection microscope, clean and dissect away the ovarian fat pad and connective tissue to allow clear visualization of the oviduct (Figure 3B).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63168/63168fig03.jpg" /> 
Figure 3: Step by step oviduct microdissection. Following removal of remnant fat and connective tissue (A,B), add toluidine blue to the tissue (C), and then wash away to facilitate visualization and subsequent removal of the ovary and uncoiling of the tube (D-H). The infundibulum can be found beside the proximal isthmus and uterine-tubal junction (UTJ). Lightly pull the distal end while cutting at the mesosalpinx to uncoil and reveal the endogenous turns of the oviduct (H) to orient for appropriate segmentation. Figure A-C scale bar = 500 &#956;m, D-H scale bar = 400 &#956;m (I) Cartoon of the various segments (not drawn to scale). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63168/63168fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Using a 1 mL syringe, dispense and incubate the oviduct affixed to the uterus in 1-2 drops of sterile 1% toluidine blue dye solution&#160;for 30 s-1 min. Rinse with cold Dulbecco&#39;s phosphate-buffered saline (DPBS) and remove all the liquid (Figure 3C).</li>
	<li>Lightly pull the ovary from the coiled oviduct and cut away at the bursal membrane and broad ligament to remove the ovary from the oviduct without compromising the tubal tissue (Figure 3D,E). 
	&#8203;NOTE: The ovary is not attached to the oviduct but encased in the bursal membrane with the oviduct (Figure 2D) and connected to the lateral side of the uterus via the broad ligament.</li>
</ol>
3. Microdissection and segmentation of the oviduct
<ol>
	<li>Locate the distal, fimbrial end of the oviduct found lateral to the uterine-tubal junction (UTJ). The oviduct coils back on itself; hence the fimbrial end can be located lateral to the proximal end and is an appropriate starting point to orient for uncoiling of the oviduct (Figure 3F,I).
	<ol>
		<li>While gently pulling the tube, cut away at the mesosalpinx (Figure 3I) with spring-form micro-scissors to uncoil the oviduct (Figure 3G).</li>
	</ol>
	</li>
	<li>Once uncoiled, cut the tube to produce the infundibular, ampullary, and isthmic regions (Figure 3H).
	<ol>
		<li>Following excision of the infundibulum (fimbrial end and proximal stalk), excise the ampullary region by cutting after the second prominent turn of the tube (cut between turns two and three, approximately 1 cm in length). Finally, cut the remaining portion to the UTJ, which is the isthmic region (Figure 3H). 
		NOTE: The oviduct will not completely uncoil into a straight structure. The tube will maintain its turns (Figure 3H)3. Based on the subsequent turns following the ampullary region, one may further segment the remaining tube into the ampullary-isthmic junction and isthmus3. Following ampulla excision, cut between turns five and six to obtain the ampullary-isthmic junction; the remaining tube (turn six to the start of the UTJ) is the isthmus3.</li>
	</ol>
	</li>
	<li>Immediately snap freeze dissected tissue segments in liquid nitrogen for RNA isolation or fix and process each segment for oriented embedding and immunohistological analysis.
	<ol>
		<li>Add 200 &#956;L of cold RNA extraction reagent and 1:1 homogenization bead mix (see Table of materials) to the tissue if proceeding for RNA extraction (see step 5.1).</li>
	</ol>
	</li>
</ol>
4. Oviduct dissociation
<ol>
	<li>Continued from step 3.1.1 above) For optimal dissociation of the epithelium, slit open the oviduct in the areas where possible (i.e., infundibulum and ampulla) to expose the luminal epithelium (Figure 4A).</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63168/63168fig04.jpg" /> 
Figure 4: Oviduct cell dissociation, part 1. Cartoon showing how to expose the distal epithelium for optimal cell dissociation (A) and an image showing the easiest way to use the 1 mm2 mesh (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63168/63168fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="1" style="margin-left: 40px;">
	<li>Starting at the fimbrial region, using forceps as leverage, slit the tube longitudinally with spring-form scissors.</li>
</ol>
<ol start="2">
	<li>Mince the slit-open portions and the remaining oviduct into segments (~1-2 mm in size) and add 5 mL of warm, non-enzymatic dissociation buffer (see Table of Materials) and incubate at 37 &#176;C.
	<ol>
		<li>Resuspend the tissue pieces and dissociated cells with a bulb pipettor every 3 min for 9-10 min. Dilute the dissociation buffer 1:1 with cold dissection medium.</li>
	</ol>
	</li>
	<li>Collect the cells by centrifugation (350 x g, 3 min, 4 &#176;C). Resuspend the cells in 2 mL of RBC lysis buffer for 2 min at room temperature, and then dilute 1:1 with cold dissection medium.</li>
	<li>Collect the cells by centrifugation (350 x g, 3 min, 4 &#176;C) and resuspend in 5 mL of cold pronase solution.
	<ol>
		<li>Incubate in pronase digestion medium and resuspend the cell clumps with a bulb pipettor every 3-5 min until a single cell suspension is achieved (~25-30 min). 
		NOTE: This step is best performed in a tissue culture plate or flask to allow constant observation preventing unnecessary overexposure to pronase.</li>
	</ol>
	</li>
	<li>Pass the cells through a sterile macro-mesh (1 mm x 1 mm mesh affixed to a conical tube with a rubber band (Figure 4B)) to remove any remaining undissociated tissue pieces. Collect the cells by centrifugation (350 x g, 3 min, 4 &#176;C) and resuspend them in a cold medium appropriate for downstream analysis (e.g., FACS buffer if proceeding with staining for flow cytometry).</li>
</ol>
5. RNA extraction of oviductal segments
<ol>
	<li>Homogenize snap-frozen samples in 200 &#181;L of RNA extraction reagent in a bead bullet blender tissue homogenizer on level 12 for two intervals (3 min each) at 4 &#176;C using the stainless-steel bead mix. 
	NOTE: To preserve RNA integrity, samples should be immediately moved from snap-frozen state into RNA extraction reagent and not weighed.</li>
	<li>Briefly centrifuge homogenate at 100-200 x g in a tabletop centrifuge for approximately 5 s at room temperature to separate the liquid from the beads. Transfer the supernatant to a fresh tube.</li>
	<li>Add another 200 &#181;L of RNA extraction reagent to the beads to recover more samples, followed by centrifugation (100-200 x g, 5 s, room temperature). Combine the supernatants. 
	NOTE: Use a small pipette tip (most p200 tips are suitable) to prevent bead carryover when removing the supernatant.</li>
	<li>Follow manufacturer&#39;s guidelines to phase separate and precipitate RNA. Representative samples were precipitated overnight at -20 &#176;C with 100% isopropanol (approximately 2:1 volume, isopropanol: recovered aqueous phase).</li>
	<li>Transfer the complete RNA precipitate along with the solution to an RNA-binding spin column and centrifuge for 30 s at 9,500 x g. Purify the RNA on-column as per the manufacturer&#39;s guidelines, and then elute the RNA in 20 &#181;L of nuclease-free water. Assess the quality/quantity on a microvolume spectrophotometer and/or bioanalyzer.</li>
</ol>

<ol>
	<li>Stewart, C. A., et al., Kubiak, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mouse oviduct developmemt in Mouse Development: From Oocyte to Stem Cells. , 247-262 (2012).</li><li>Ford, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oviduct+epithelial+cells+constitute+two+developmentally+distinct+lineages+that+are+spatially+separated+along+the+distal-proximal+axis.">Oviduct epithelial cells constitute two developmentally distinct lineages that are spatially separated along the distal-proximal axis.</a> Cell Reports. 36 (10), 109677 (2021).</li><li>Harwalkar, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+and+cellular+heterogeneity+in+the+mouse+oviduct-its+potential+roles+in+reproduction+and+preimplantation+development.">Anatomical and cellular heterogeneity in the mouse oviduct-its potential roles in reproduction and preimplantation development.</a> Biology of Reproduction. 104 (6), 1249-1261 (2021).</li><li>Agduhr, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+structure+and+development+of+the+bursa+ovarica+and+the+tuba+uterina+in+the+mouse.">Studies on the structure and development of the bursa ovarica and the tuba uterina in the mouse.</a> Acta Zoologica. 8 (1), 1 (1927).</li><li>Pulkkinen, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oviductal+function+is+critical+for+very+early+human+life.">Oviductal function is critical for very early human life.</a> Annals of Medicine. 27 (3), 307-310 (1995).</li><li>Kindelberger, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraepithelial+carcinoma+of+the+fimbria+and+pelvic+serous+carcinoma:+Evidence+for+a+causal+relationship.">Intraepithelial carcinoma of the fimbria and pelvic serous carcinoma: Evidence for a causal relationship.</a> American Journal of Surgical Pathology. 31 (2), 161-169 (2007).</li><li>Ghosh, A., Syed, S. M., Tanwar, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genetic+cell+lineage+tracing+reveals+that+oviductal+secretory+cells+self-renew+and+give+rise+to+ciliated+cells.">In vivo genetic cell lineage tracing reveals that oviductal secretory cells self-renew and give rise to ciliated cells.</a> Development. 144 (17), 3031-3041 (2017).</li><li>Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+candidate+precursor+to+serous+carcinoma+that+originates+in+the+distal+fallopian+tube.">A candidate precursor to serous carcinoma that originates in the distal fallopian tube.</a> Journal of Pathology. 211 (1), 26-35 (2007).</li><li>Kim, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-grade+serous+ovarian+cancer+arises+from+fallopian+tube+in+a+mouse+model.">High-grade serous ovarian cancer arises from fallopian tube in a mouse model.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (10), 3921-3926 (2012).</li><li>Perets, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+the+fallopian+tube+secretory+epithelium+leads+to+high-grade+serous+ovarian+cancer+in+Brca;Tp53;Pten+models.">Transformation of the fallopian tube secretory epithelium leads to high-grade serous ovarian cancer in Brca;Tp53;Pten models.</a> Cancer Cell. 24 (6), 751-765 (2013).</li><li>Sherman-Baust, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+engineered+ovarian+cancer+mouse+model+based+on+fallopian+tube+transformation+mimics+human+high-grade+serous+carcinoma+development.">A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high-grade serous carcinoma development.</a> Journal of Pathology. 233 (3), 228-237 (2014).</li><li>Zhai, Y. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-grade+serous+carcinomas+arise+in+the+mouse+oviduct+via+defects+linked+to+the+human+disease.">High-grade serous carcinomas arise in the mouse oviduct via defects linked to the human disease.</a> Journal of Pathology. 243 (1), 16-25 (2017).</li><li>Karthikeyan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolactin+signaling+drives+tumorigenesis+in+human+high+grade+serous+ovarian+cancer+cells+and+in+a+spontaneous+fallopian+tube+derived+model.">Prolactin signaling drives tumorigenesis in human high grade serous ovarian cancer cells and in a spontaneous fallopian tube derived model.</a> Cancer Letters. 433, 221-231 (2018).</li><li>Shao, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+prolactin+receptor+(PRLR)+in+mouse+and+human+fallopian+tubes:+Evidence+for+multiple+regulatory+mechanisms+controlling+PRLR+isoform+expression+in+mice.">Differences in prolactin receptor (PRLR) in mouse and human fallopian tubes: Evidence for multiple regulatory mechanisms controlling PRLR isoform expression in mice.</a> Biology of Reproduction. 79 (4), 748-757 (2008).</li><li>Alwosaibai, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAX2+maintains+the+differentiation+of+mouse+oviductal+epithelium+and+inhibits+the+transition+to+a+stem+cell-like+state.">PAX2 maintains the differentiation of mouse oviductal epithelium and inhibits the transition to a stem cell-like state.</a> Oncotarget. 8 (44), 76881-76897 (2017).</li><li>Peri, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+class+of+interstitial+cells+in+the+mouse+and+monkey+female+reproductive+tracts.">A novel class of interstitial cells in the mouse and monkey female reproductive tracts.</a> Biology of Reproduction. 92 (4), 102 (2015).</li><li>L&#245;hmussaar, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+origin+of+high-grade+serous+ovarian+cancer+using+CRISPR-modification+of+mouse+organoids.">Assessing the origin of high-grade serous ovarian cancer using CRISPR-modification of mouse organoids.</a> Nature Communications. 11 (1), 2660 (2020).</li><li>Chen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+air-liquid+interphase+approach+for+modeling+the+early+embryo-maternal+contact+zone.">An air-liquid interphase approach for modeling the early embryo-maternal contact zone.</a> Scientific Reports. 7, 42298 (2017).</li><li>Desjardins, P., Conklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoDrop+microvolume+quantitation+of+nucleic+acids.">NanoDrop microvolume quantitation of nucleic acids.</a> Journal of Visualized Experiments: JoVE. (45), e2565 (2010).</li><li>Schroeder, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Molecular Biology. 7, 3 (2006).</li><li>McGlade, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type+specific+analysis+of+physiological+action+of+estrogen+in+mouse+oviducts.">Cell-type specific analysis of physiological action of estrogen in mouse oviducts.</a> The FASEB Journal. 35 (5), 21563 (2021).</li></ol>

The authors have nothing to disclose.

The three segments of the oviduct are histologically, morphologically, and functionally distinct1,2,3. The epithelium varies greatly from one end of the oviduct to the other. Ciliated cells dominate at the fimbrial/infundibular end, while secretory cells dominate in the isthmic region1. While this overall gradient has been recognized for some time, recent work has uncovered more distinctions among the oviductal segments. Thus, while ciliated cells become less frequent toward the proximal end of the tube, there is a sharp drop in their number (from approximately 60% to 10% ciliation) transitioning from the ampulla to the ampullary-isthmic junction3. Further, epithelial cell populations in the proximal and distal segments are derived from developmentally distinct lineages1,2,3. Distal epithelial cells of the infundibulum and ampulla do not contribute to proximal epithelial cell populations in the isthmus, as shown by lineage tracing of epithelial markers. At embryonic day 12.5 (E12.5), oviductal epithelial cells have a distinct lineage independent of the proximal epithelial cell marker, PAX22. These discoveries, in addition to the diverse cell population among the various segments, further emphasize the necessity to investigate the segments independently. The described method details a step-by-step unwinding and segment identification protocol as per the three historically described segments (infundibulum, ampulla, and isthmus). Due to the recent establishment of the ampullary-isthmic junction as a definitive oviduct segment, priority was given to the identification and collection of the three classically described areas. However, the method can be extended to include a separate collection of the ampullary-isthmic junction, as described above3.
The use of the blue dye and location of the infundibulum is a critical step to unwind the oviduct in a fast and efficient way. The blue dye highlights the ostium and coils of the tube, allowing a faster and more efficient de-coiling and collection. The initial location of the ostium orients one to the distal (infundibular) and proximal (isthmic) areas of the oviduct throughout uncoiling. In addition, keeping the proximal uterine horn portion affixed to the dissection platform during uncoiling allows the surgeon to use both hands for the delicate dissection steps. Initial removal of the coiled oviduct and attempts to uncoil this structure without rendering the tube immobile or without the use of the dye can easily lead to confusion regarding the distal and proximal ends or cause breakage of the tube. Tube breakage is easy, so care must be exercised when uncoiling since breakage will make reproducible segmentation unachievable. The use of high-quality, spring-form micro-scissors is highly recommended to achieve the best results.
Evaluating the gene expression signature in the diverse segments of the oviduct, for example, during the estrous cycle and in response to administered hormones and cytokines, will most likely produce a much better understanding of how the oviduct changes in response to these stimuli. These data may also shed light on what factors contribute to epithelial transformation. The RNA extraction protocol herein was developed to obtain the highest quantity and quality possible for this tissue of limited size and resilient tubal structure. The described homogenization, RNA precipitation, and conjunctional use of the phenol: chloroform and on-column purification methods were found to be most efficient in achieving appropriate quality and quantity of RNA for downstream analysis. The oviductal segments were not weighed and entire segments were immediately processed for tissue homogenization in order to prevent any warming of the tissue leading to RNA degradation. In addition, because the small tubal segments require multiple homogenization steps, it is critical to ensure that homogenization is conducted at 4 &#176;C to preserve sample integrity. Because this method is sufficient to produce appropriate yield for downstream analysis, a future application of this technique would be to perform segmental RNAseq, previously not reported, which would contribute greatly to our understanding of oviduct physiology.
During the development of the cell dissociation protocol, we found that robust and/or long enzymatic digestions caused demonstrable loss of cilia. We, therefore, concentrated on the development of, and achieved, a method that preserved cell morphology. Improvements in single cell yield, as described herein, will allow more robust and immediate multiplexed analyses of the different epithelial and stromal cell types by flow cytometry. The use of a non-enzymatic dissociation protocol followed by a short incubation in pronase is also likely to improve the preservation of cell surface antigens over dissociation methods utilizing long or robust digestion methods2,18. Single cell sequencing analysis requires far fewer cells compared to multiplexed flow cytometry panels2,21. So single cell sequencing of the three described segments of the oviduct may be possible with the described protocol.
Because the oviduct cannot be fileted along its entire length, a potential limitation of the current method could be that cells forming the epithelium of the narrower isthmus would have reduced exposure to the dissociation reagents and, therefore, may not be present in the final suspension in the same proportion as in vivo.
Due to the coiling of the oviduct, orientation for immunohistochemical analyses of all oviductal segments can be challenging3. However, the described segment dissection followed by oriented embedding of the oviductal segments allows for more comprehensive longitudinal and cross-sectional image analysis without having to serially section through the entire intact coiled tube. Further, the blue dye utilized for uncoiling, aids in correct orientation during embedment of the small tubal segments.

The murine oviduct is similar in function and morphology to the human fallopian tube1. Both consist of a pseudostratified ciliated epithelium, consisting of two historically described epithelial resident cells: ciliated cells and secretory cells1,2. The oviduct has three classically recognized segments: the infundibulum, the ampulla, and the isthmus. In a recent study, Harwalkar et al.3 investigated oviduct morphology and gene expression leading to the expansion of the categorization of resident epithelial cells to seven distinct populations. In addition, they established the ampullary-isthmus junction as a distinct segment of the oviduct3. The method described herein, which focuses on the infundibulum, ampulla, and isthmus, could easily be extended to include the ampullary-isthmic junction as well2,3. The infundibular region contains the ostium, or opening of the oviduct, and includes the fimbrial region as well as the proximal stalk. Moving toward the uterus, next is the ampulla, and then the isthmus. Ciliated cells are most prominent in the distal end of the region, proximal to the ovary, or infundibulum, while secretory cells are most prominent in the proximal end or isthmus segment1. Unlike the human fallopian tube, the murine oviduct is a coiled structure supported by the mesosalpinx, an extension of the broad ligament peritoneum1,4. In addition, the mouse oviduct is encased in a bursal sac that increases the likelihood of oocyte transfer into the oviduct4. The ampulla is identified as the location of fertilization, from which developing embryos pass into the isthmus before entering the uterus5. Tubal segments are 200-400 &#956;m in diameter and the longer ampullary and isthmus regions are approximately 0.5-1.0 cm in length4. The oviduct distends during the estrous cycle and the ampulla and infundibulum are more distensible than the isthmus1.
Over proliferation of cells, especially secretory cells, characterize precursor lesions to serous tumors found in the pelvic cavity6. These precursor serous intraepithelial lesions arise in the oviduct epithelium solely in the fimbrial region; it is unknown why lesion formation is restricted to this region where normally the predominant cell type is ciliated, not secretory2,7,8. The regionality in terms of normal physiological function, as well as heightened interest in the oviductal origin of ovarian cancer9,10,11,12,13, underscores the importance of separate evaluation of the oviduct segments.
The method described here details the collection of separate oviductal segments for subsequent downstream analyses of segment-specific gene expression and function of dissociated cells. Traditionally, many tissues are processed for whole RNA extraction following either the phenol: chloroform method or an on-column complete extraction method; however, we found that RNA quality was maintained while producing sufficient yield with the described combination method. Utilizing this method, very small functional segments of the oviduct can be processed for downstream analyses rather than investigating the oviduct as a whole, which can mask results representative of the different segments14.
Dissociated murine oviductal cells have rarely been investigated by flow cytometry, most likely due to the limiting cell yield from this tissue. One approach to overcome this problem has been to dissociate cells, grow them in culture, and then stimulate re-differentiation in vitro to obtain appropriate cell numbers for downstream cell analysis15,16,17,18. A limitation to this approach is the time ex vivo and altered microenvironment in culture, both of which likely change gene expression. There is also an assumption that morphological re-differentiation has the same transcriptional and proteomic signature as was present in the intact animal. The current dissociation method was designed to achieve the highest number of epithelial cells in a heterogenous oviductal cell population while maintaining single cell differentiation. Further, the mostly non-enzymatic approach likely limits the loss of cell surface proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 mm Stainless steel bead mix</td>
			<td>Next Advance</td>
			<td>SSB05</td>
			<td>Mix 1:1 with 1.4 mm SSB14B, sterilized</td>
		</tr>
		<tr>
			<td>1.4 mm Stainless steel bead mix</td>
			<td>Next Advance</td>
			<td>SSB14B</td>
			<td>Mix 1:1 with 0.5 mm SSB05, sterilized</td>
		</tr>
		<tr>
			<td>1X Dulbecco&#39;s Phosphate Buffered Saline A, pH 7.4 (DPBS)</td>
			<td>Gibco</td>
			<td>21600-010</td>
			<td>Cold, sterile</td>
		</tr>
		<tr>
			<td>25G needle</td>
			<td>BD</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm sterile petri dishes</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell meshes</td>
			<td>Fisherbrand</td>
			<td>22-636-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent Eukaryote Total RNA 6000 Pico Chip kit</td>
			<td>Agilent 2100 Bioanalyzer</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead Bullet Blender Tissue Homogenizer</td>
			<td>Next Advance</td>
			<td>BBY24M</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanlyzer</td>
			<td>Agilent 2100 Bioanalyzer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cold plate/pack/surface of choice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Kept at -20C for dissection</td>
		</tr>
		<tr>
			<td>Dental wax</td>
			<td>Polysciences Inc.</td>
			<td>403</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/ Ham&#8217;s F12</td>
			<td>Corning</td>
			<td>10-090-CV</td>
			<td>Prepare dissection medium: DMEM/ Ham&#8217;s F12, 10% FBS, 25 mM Hepes, 1% Pen-Strep</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-015CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine point forceps of choice</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma Aldrich</td>
			<td>G712b</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A-21422</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11001</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma Aldrich</td>
			<td>H-3784</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht 33342</td>
			<td>Cell Signaling Technologies</td>
			<td>4082S</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Inverted compound microscope</td>
			<td>Keyence BZ-X700</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-mouse Occludin</td>
			<td>Invitrogen</td>
			<td>33-1500</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Non-enzymatic dissociation buffer</td>
			<td>N/A</td>
			<td></td>
			<td>5 mM EDTA, 1 g/L glucose, 0.4% BSA, 1X DPBS</td>
		</tr>
		<tr>
			<td>Nylon macro-mesh 1 mm x 1 mm</td>
			<td>Thomas Scientific</td>
			<td>1210U04</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P-6148</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>MP Biomedicals</td>
			<td>10220-718</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent</td>
			<td>Cell Signaling Technologies</td>
			<td>9071S</td>
			<td>Immunocytochemical validation images: antifade mounting medium</td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma Aldrich</td>
			<td>10165921001</td>
			<td>Prepare pronase digestion medium: 0.15% Pronase in DMEM/Ham&#39;s F12, sterile</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Roche</td>
			<td>11 348 639 001</td>
			<td>Viability validation images</td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse Acetylated-Tubulin</td>
			<td>Abcam</td>
			<td>ab179484</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td>Utilized for on-column purification in text.</td>
		</tr>
		<tr>
			<td>Spring form microdissection scissors</td>
			<td>Roboz Surgical</td>
			<td>RS-5610</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 3 mL bulb pipettors</td>
			<td>Globe Scientific</td>
			<td>137135</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue</td>
			<td>Alfa Aesar</td>
			<td>J66015</td>
			<td>Prepare toluidine blue solution: 1% in 1X DPBS, sterile</td>
		</tr>
		<tr>
			<td>Tris-Buffered Saline-Tween (TBST)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Immunocytochemical validation images; 0.1 N NaCl, 10 mM Tris-Cl pH 7.5, 1% Tween 20</td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Mallinckrodt Inc.</td>
			<td>3555</td>
			<td>Immunocytochemical validation images</td>
		</tr>
		<tr>
			<td>Trizol RNA Extraction Reagent</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td>Referred to as RNA extraction reagent. Nucelase-free water, chloroform and isoproponal are required in supplement of performing Trizol extraction per manufacturer&#39;s guidelines</td>
		</tr>
	</tbody>
</table>

microdissection and dissociation of the murine oviduct: individual segment identification and single cell isolation

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However, because of their small body size, some structures, such as the oviduct with a diameter of 200-400 &#956;m, have proven to be relatively difficult to study except by immunohistochemistry. Recently, immunohistochemical studies have uncovered more complex differences in oviduct segments than were previously recognized; thus, the oviduct is divided into four functional segments with different ratios of seven distinct epithelial cell types. The different embryological origins and ratios of the epithelial cell types likely make the four functional regions differentially susceptible to disease. For example, precursor lesions to serous intraepithelial carcinomas arise from the infundibulum in mouse models and from the corresponding fimbrial region in the human fallopian tube. The protocol described here details a method for microdissection to subdivide the oviduct in such a way to yield a sufficient amount and purity of RNA necessary for downstream analysis such as reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing (RNAseq). Also described is a mostly non-enzymatic tissue dissociation method appropriate for flow cytometry or single cell RNAseq analysis of fully differentiated oviductal cells. The methods described will facilitate further research utilizing the murine oviduct in the field of reproduction, fertility, cancer, and immunology.

The described dissociation protocol yields 100,000-120,000 cells per mouse with pooling of both oviducts. The method is gentle enough to leave multi-ciliated cell borders intact, allowing for a distinction between multi-ciliated cells and secretory cells, and verifying that the digestion method is gentle enough to prevent de-differentiation. Representative immunofluorescence images in Figure 5 show small cell clumps following step 4.2.1, fixed for 3 min in 4% paraformaldehyde (PFA)/ DPBS, washed with 1% bovine serum albumin (BSA)/ Tris-buffered saline-tween (BTBST) and stained in suspension. Briefly, cells were permeabilized for 15 min at room temperature in 0.5% TritonX-100/BTBST, washed in BTBST, quenched for background fluorescence for 2 h at room temperature in 20 mM glycine/ DPBS, washed in BTBST, and then blocked for 1 h at room temperature in 5% BSA/ 0.1% TritonX-100/ TBST. Cells were then incubated in the primary antibody at 4 &#176;C overnight in BTBST (mouse anti-mouse occludin (1:2000); rabbit anti-mouse acetylated tubulin (1:1000)) followed by several washes in BTBST. Secondary antibody was added for 1 h at room temperature in BTBST (goat anti-mouse IgG Alexa Fluor 555 (1:1000); goat anti-rabbit IgG Alexa Fluor 488 (1:1000)), washed several times in BTBST, once in DPBS, and then incubated in nuclear stain for 20 min at room temperature (1:2000, Hoescht 33342) followed by a wash in DPBS. Cells were resuspended in 80 &#181;L of antifade mounting medium and left overnight in the dark before imaging (Figure 5A). Figure 5B shows cells at the end of the dissociation protocol. Viability assessments were made throughout the protocol, and representative images are shown in Figure 5B-D. Propidium iodide was added at 1:100 dilution, and cells were immediately observed on an inverted compound fluorescence microscope (Figure 5C, red stain). Cilia remained intact in small cell clusters (Figure 5A) and single cell suspensions (Figure 5E) with many cells observed to have cilia still beating (Suppl. Video 1).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63168/63168fig05.jpg" /> 
Figure 5: Oviduct cell dissociation, part 2. Fluorescent and phase-contrast images of a cell cluster positive for occludin (red), nuclei (blue), and acetylated tubulin (green). The ciliated apical border (green in AI and AV) remains intact throughout the protocol and withstands further digestion to single cells (E). AI: merge, AII: red channel, AIII: blue channel, AIV: phase contrast, AV: green channel. After a brief pronase incubation, the majority of cells are single. Propidium iodide (PI) staining shows approximately 93% viability. (B) Bright field, (C) red channel PI positive, (D) merge. (E) inset on merge shows an intact ciliated border on a single dissociated cell. Such cells had actively beating cilia (see Suppl. Video 1). Images were taken on an inverted compound fluorescence microscope. Figure AI-V scale bar = 20 &#956;m, B-D scale bar = 50 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63168/63168fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The RNA isolation protocol described gives sufficient yield and purity necessary for segmental RT-qPCR and RNAseq. Results show that this method yields 800-1200 ng RNA per segment (Figure 6A) except for infundibular samples where pooling from two animals may be necessary for appropriate yield for downstream assays. The presented results for infundibulum are pooled from two animals, totaling four infundibular regions (Figure 6). This protocol is successful in achieving pure RNA, initially analyzed by microvolume spectrophotometer and further by bioanalyzer (Figure 6B,C). Samples have a 260/280 nm absorption ratio between 1.8 and 2.0 (Figure 6B) typical of pure RNA nucleotide species19. Absorption ratios (260/230 nm) were taken for each sample with an inclusion criterion between 1 and 2.2, representative of limited contamination from isolation reagents (data not shown)19. Bioanalyzing the samples showed high integrity of the RNA, with assessed RNA integrity number (RIN) above seven (Figure 6C), appropriate for downstream expression and sequencing analysis20.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63168/63168fig06.jpg" /> 
Figure 6: Quality assessment of segmental RNA. Whole RNA was assessed for yield (A) and quality (B,C) by microvolume spectrophotometer and bioanalyzer. RIN: RNA integrity number, as assessed by the bioanalyzer. Bars are the mean &#177; standard deviation (SD), N = 12 samples per segment from two separate RNA extractions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63168/63168fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Video 1: Live cell imaging of beating cilia on dissociated cells. Beating cilia following pronase digestion. Videos were taken on an inverted compound microscope. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63168/pi_2.avi" target="_blank">Please click here to download this Video.</a>

Watch this Scientific Journal Video about Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation at JoVE.com

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental ...

microdissection-dissociation-murine-oviduct-individual-segment

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

3.7K Views.  University of California, Riverside. A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.

Video: Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined &#8220;middle&#8221; of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.

This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Cell Dissociation from the Tongue Epithelium and Mesenchyme/Connective Tissue of Embryonic-Day 12.5 and 8-Week-Old Mice

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 &#176;C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (&#62;90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.

We have developed a generalized protocol to dissociate a large quantity of high-quality single cells from the epithelium and mesenchyme/connective tissue of embryonic and adult mouse tongues.

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g.,&#160;single cell RNA ...

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...