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="Oviduct dissection stages; includes dye application and ovary removal; anatomical diagram; microscope." class="xfigimg" src="/files/ftp_upload/63168/63168fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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 °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="Reproductive anatomy setup; diagram and image show ovary, oviduct, and uterus with labeling details." class="xfigimg" src="/files/ftp_upload/63168/63168fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
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="Dissection process of mouse oviduct; microscopy images with labeled anatomical structures." class="xfigimg" src="/files/ftp_upload/63168/63168fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
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 μm, D-H scale bar = 400 μm (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 for 30 s-1 min. Rinse with cold Dulbecco'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). 
	​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 μL 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="Oviduct anatomy diagram and epithelium culture method with lab jar setup for reproductive research." class="xfigimg" src="/files/ftp_upload/63168/63168fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
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 °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 °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 °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 °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 µL of RNA extraction reagent in a bead bullet blender tissue homogenizer on level 12 for two intervals (3 min each) at 4 °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 µL 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's guidelines to phase separate and precipitate RNA. Representative samples were precipitated overnight at -20 °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's guidelines, and then elute the RNA in 20 µL of nuclease-free water. Assess the quality/quantity on a microvolume spectrophotometer and/or bioanalyzer.</li>
</ol>

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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 ovi...

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><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&#039;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 &amp;mu;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 -20 &amp;#176;C for dissection</td></tr><tr><td>Dental wax</td><td>Polysciences Inc.</td><td>403</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)/ Ham&amp;rsquo;s F12</td><td>Corning</td><td>10-090-CV</td><td>Prepare dissection medium: DMEM/ Ham&amp;rsquo;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&#039;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&#039;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, wa...

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

Research

JoVE Journal

Developmental Biology

3.8K 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

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

Cancer Research

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Immunology and Infection

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 ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Neuroscience

Semi-Automated Mechanical-Enzymatic Tissue Dissociation for Single-Cell Analysis

Mechanical Dissociation of Tissues for Single Cell Analysis Using a Motorized Device

Being able to isolate and prepare single cells for the analysis of tissue samples has rapidly become crucial for new biomedical discoveries and research. Manual protocols for single-cell isolations are highly time-consuming and prone to user variability. Automated mechanical protocols are able to reduce processing time and sample variability but aren't easily accessible or cost-effective in lower-resourced research settings. The device described here was designed for semi-automated tissue dissociation using commercially available materials as a low-cost alternative for academic laboratories. Instructions to fabricate, assemble, and operate the device design have been provided. The dissociation protocol reliably produces single-cell suspensions with comparable cell yields and sample viability to manual preparations across multiple mouse tissues. The protocol provides the ability to process up to 12 tissue samples simultaneously per device, making studies requiring large sample sizes more manageable. The accompanying software also allows for customization of the device protocol to accommodate varying tissues and experimental constraints.

Author Spotlight: Advancements in Nanoparticle Technology for Drug Delivery and Immunotherapy

A general protocol for the combined enzymatic and semi-automated mechanical dissociation of tissues to generate single-cell suspensions for downstream analyses, such as flow cytometry, is provided. Instructions for the fabrication, assembly, and operation of the low-cost mechanical device developed for this protocol are included.

Being able to isolate and prepare single cells for the analysis of tissue samples has rapidly become crucial for new biomedical discoveries and research. ...

Bioengineering

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Biology

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

Medicine

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...