Authors Ruegg L, James-Allan LB and DiBernardo G are partially supported by the Greater Los Angeles Veterans Association projects 1I01BX006019-01A2 and I01BX006411-01 to Memarzadeh&#160;S. Author Ochoa C is supported by UCLA Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research Rose Hills Foundation Graduate Scholarship Training Program. We want to thank the Translational Pathology Core Laboratory at UCLA and specifically Ko Kiehle and Chloe Yin for assistance in tissue procurement. We also want to thank Ken Yamauchi and the BSCRC Microscopy core for their assistance in imaging. Figure 1 was created with BioRender.com (agreement number JL27QWDYNT). Lastly, we would like to thank Felicia Cordea and the BSCRC flow cytometry core for assistance in flow cytometry.

This protocol was adapted from a uterine epithelial cell isolation method described previously13,14. Fresh specimens of deidentified fallopian tubes were collected through our University of California, Los Angeles (UCLA) IRB-approved protocol #10-0727 and digested into a single-cell suspension within approximately 4&#8211;6 h.
1. Fallopian tube collection and preparation 
<ol>
	<li>Collect fresh human fallopian tubes (Figure 1A) in dissociation media (DMEM [4.5 g/L D-glucose] containing 10% fetal bovine serum (FBS), 5 mL L-glutamine, and 5 mL of penicillin/streptomycin) in 15 mL or 50 mL tubes and transport&#160;on ice to the laboratory.</li>
	<li>Transfer the fallopian tubes into a sterile Petri dish containing fresh dissociation media.&#160;&#160;</li>
	<li>Under a dissecting microscope, dissect away fat, connective tissue, and any vessels surrounding the tubes using fine point forceps and Vannas Tubingen scissors (Figure 1B).</li>
	<li>Under the dissecting microscope, cut through the coronal plane of fallopian tubes with scissors to form small cylinders, ~3&#8211;5 mm each in diameter (Figure 1C), using the fine point forceps to stabilize the tissue.</li>
</ol>
2. Epithelial cell isolation and dissociation
<ol>
	<li>Carefully aspirate the dissociation media from the dish.</li>
	<li>Wash the tissue pieces 2x with cold 1x phosphate-buffered saline (PBS) by gently pipetting ~2 mL to cover the pieces and lightly rotating the plate.</li>
	<li>Aspirate the final 1x PBS wash, incubate the tissue pieces for 5 min in cold 5 mM ethylenediaminetetraacetic acid (EDTA) at room temperature, and repeat for a total of two incubations. Aspirate the 5mM EDTA after each incubation.</li>
	<li>Suspend the tissue fragments in cold 1% Trypsin/Hanks&#39; Balanced Salt Solution (HBSS) and incubate at 4 &#730;C for 40 min (Figure 1D).</li>
	<li>Aspirate the 1% trypsin/HBSS and wash the tissue pieces 2x in cold dissociation media to inactivate the trypsin.</li>
	<li>Incubate the tissue fragments in 2 mL of dissociation media with 0.4 mg of DNase for at least 30 min at room temperature. 
	NOTE: A longer incubation period may be necessary, but do not exceed 45 min before starting step 2.7.</li>
	<li>Under a dissecting microscope, while still in the media with DNase, hold a fragment of the fallopian tube, the lumen parallel to the bottom of the dish, in place with forceps. With the other hand, repeatedly press on the fallopian tube piece with forceps to expel epithelial cells from the lumen of the tube. Confirm that a halo of cells is seen around the tissue as cells are released from the lumen of the tube. Repeat for each fragment of fallopian tube in the dish (Figure 1E).</li>
	<li>Transfer the media containing cells into a sterile 15 mL tube, wash the Petri dish with dissociation media at least 2x, and combine the washes into the 15 mL tube. Discard the stromal tube fragments.</li>
	<li>Harvest cells by centrifugation at 500 &#215; g for 5 min. Aspirate the supernatant and resuspend the pellet in 1 mL of dissociation media.</li>
	<li>Count cells by aliquoting 10 &#181;L of the cell suspension and mixing 1:1 with trypan blue. Pipette 10 &#181;L of the mixture into a chamber counting slide and insert it into a cell counter (Figure 1F).</li>
</ol>
3. Digestion to single-cell suspension 
<ol>
	<li>Resuspend the cells in 9 mL of dissociation media, 1 mL of 8 mg/mL collagenase (type 1), and 0.4 mg of DNase. Incubate at 37 &#730;C for 30&#8211;45 min in an orbital shaker at 200&#160;rpm (Figure 1G).</li>
	<li>Harvest the cells by centrifugation at 500 &#215; g for 5 min. Aspirate the supernatant and resuspend the pellet in 5 mL of dissociation media to wash off collagenase.</li>
	<li>Pass the cell suspension through a 100 &#181;m cell strainer into a 50 mL tube (Figure 1H).</li>
	<li>Harvest the cells by centrifugation at 500 &#215; g for 5 min.</li>
	<li>If the cell pellet appears red, carry out red blood cell (RBC) lysis as described in steps 3.6&#8211;3.8. If the cell pellet is not red, skip to step 3.9.</li>
	<li>Dilute 500 &#181;L of RBC lysis buffer in 4.5 mL of ultrapure water.</li>
	<li>Aspirate the media, add 5 mL of the diluted RBC lysis buffer, and incubate on ice for 3 min.</li>
	<li>To stop RBC lysis, add 45 mL of 1x PBS to the 50 mL tube.</li>
	<li>Harvest the cells by centrifugation at 500 &#215; g for 5 min.</li>
	<li>Count the cells by aliquoting 10 &#181;L of the cell suspension and mixing it 1:1 with trypan blue. Pipette 10 &#181;L of the mixture into a chamber counting slide and insert it into the cell counter (Figure 1I).</li>
</ol>
4. Flow cytometry staining
<ol>
	<li>Aliquot 250,000 cells into two separate 5 mL polystyrene round bottom tubes for primary conjugated antibody and isotype control to use for surface marker flow cytometry. 
	NOTE: Fewer cells can be used to accommodate for the cell count and number of cells needed for downstream analysis. To achieve optimal flow results, a minimum of 50,000 cells should be included in the analysis.</li>
	<li>Dilute the antibody (e.g., anti-human EpCAM and CD10) and isotype according to the manufacturer&#8217;s recommended dilution in 1x PBS. Include fixable viability dye (e506) and a conjugated anti-human CD45 antibody to gate out live human immune cells. Make enough antibody mixture for at least 50 &#181;L per tube. Leave in the dark on ice until step 4.4.</li>
	<li>Wash the cells with 1 mL of 1x PBS. Harvest the cells by centrifugation at 500 &#215; g for 5 min at 4 &#730;C.</li>
	<li>Aspirate the 1x PBS. Resuspend the pellet in the antibody or isotype panel and incubate at 4 &#730;C for 15 min in the dark.</li>
	<li>After incubation, dilute in 1 mL of 1x PBS and harvest the cells by centrifugation at 500 &#215; g for 5 min at 4 &#730;C.</li>
	<li>Aspirate the 1x PBS and resuspend the cell pellet in 200 &#181;L of 1x PBS.</li>
</ol>
5. Flow cytometry data collection
<ol>
	<li>Log into the FACS machine. 
	NOTE: We present the protocol utilizing the referenced flow cytometer (see the Table of Materials). For other flow cytometers, adhere to the manufacturer&#39;s instructions. The voltage setting of the flow cytometer has been preconfigured for optimal performance with human PBMCs, eliminating the need for voltage adjustments before each FACS run.</li>
	<li>If the machine is in acquisition mode, skip to step 5.3. If the machine is in data analysis mode, change to acquisition mode by pressing the power button in the top right. When a popup displays, press the button that reads acquisition mode.</li>
	<li>In the bottom left, if the machine reads Calibration Required, then proceed to step 5.3.1. If the machine reads Calibration ok, skip to step 5.4.
	<ol>
		<li>To calibrate the machine, take the calibration beads out of the 4 &#176;C fridge and vortex.</li>
		<li>Place 1 drop of calibration beads in a 5 mL polystyrene round bottom tube and place the tube in the single tube holder.</li>
		<li>Press the barcode button in the top right and scan the calibration bead bottle. A pop-up will appear. If everything is set up, click Yes | OK and let the calibration run.</li>
		<li>If the calibration is successful, log out and log back into acquisition mode. Proceed to step 5.4.</li>
	</ol>
	</li>
	<li>Click open | new workspace in top left.</li>
	<li>Select chill 5 rack; select three of the wells. Select the first well and give it a description (e.g., FTX Ab). Select the next well and give it a unique description (e.g., FTX Iso). Select the last well and give it the description bleach. 
	NOTE: Always include a bleach sample after every 4&#8211;5 experimental samples to ensure timely cleaning of the cell samples.</li>
	<li>Select all the wells and set up the experimental conditions as follows: instrument settings: Human PBMCs; Flow rate: high; Mix sample: gentle mixing; Mode: standard; Sample total volume 200 &#181;L; Sample uptake 150 &#181;L; Annotation: input antibodies and their fluorophore labeling.</li>
	<li>Select edit | options and give the file a name under description| apply | ok.</li>
	<li>Take the rack out of the fridge and place it on the tray with the barcode facing the machine. Place the sample tubes in the rack to reflect the order of samples on the screen and press the play button to start the experiment.</li>
	<li>Once the experiment has finished, plug in the USB, select File | copy, expand the private folder, select the experiment, and click copy and eject.</li>
	<li>After data collection, use software to analyze the data sheet.</li>
	<li>Start with the isotype control. Use the polygon tool to select cells on the forward scatter (FSC) versus side scatter (SSC) plot.</li>
	<li>Gate out dead cells by changing the Y-axis to e506 and the X-axis to FSC. Use the polygon tool to select the e506-negative live cell population.</li>
	<li>Double-click on the selected population. Change the Y-axis to human CD45. Use the polygon tool to select the cell population. This is the human CD45-negative cell population.</li>
	<li>Double-click on the CD45-negative cell population. Change the Y axis to histogram and X&#160;axis to human EpCAM. Use the range selection tool to click on the end of the histogram and select the empty space until the end of the graph. This represents the EpCAM-positive population. Repeat for human CD10 stain.</li>
	<li>Drag isotype gating under the antibody workspace. CD45-positive cells will be gated out and EpCAM-positive and CD10-positive populations will be selected for.</li>
	<li>To create representative histograms, select layout editor. Drag the desired histograms to the layout page. To compare isotype and antibody staining, overlay the isotype histogram with the antibody histogram. Gate the positive cells based on the isotype control.</li>
</ol>
6. Immunocytochemistry
<ol>
	<li>Culture cells after the final isolation step in the desired chamber glass slide until confluent.</li>
	<li>To fix the cells, add 500 &#181;L of cold 4% Paraformaldehyde (PFA) Incubate at room temperature for 10 min.</li>
	<li>Aspirate the 4% PFA and wash 3x with 1x PBS.</li>
	<li>To permeabilize the cells, add at least 200 &#181;L of 0.25% Triton-X in PBS and incubate at room temperature for 10 min.</li>
	<li>During permeabilization, prepare the blocking buffer (4% normal goat serum [NGS] for antibodies not produced in goat).</li>
	<li>Aspirate the 0.25% Triton-X and wash 3x with 1x PBS.</li>
	<li>Add at least 200 &#181;L of blocking buffer (4% NGS in PBS). Block for 1 h at room temperature.</li>
	<li>Prepare primary antibody dilutions in 1% NGS in 1x PBS according to the manufacturer&#8217;s recommendations.</li>
	<li>After blocking, add at least 200 &#181;L of the primary antibody solution. Incubate at room temperature for 2 h. 
	NOTE: Some primary antibodies may prefer a 4 &#176;C overnight incubation; check antibody data sheet.</li>
	<li>Prepare secondary antibody solutions in 1% NGS in 1x PBS. Wrap the tubes in tin foil and prepare solutions in the dark as the secondary antibodies are light-sensitive. Store in the dark until ready.</li>
	<li>After primary antibody incubation, wash 3x with 1x PBS.</li>
	<li>Add at least 200 &#181;L of the secondary antibody solution. Incubate in the dark at room temperature for 1 h. 
	NOTE: During the following steps, try to keep the slide&#160;in the dark as much as possible.</li>
	<li>After secondary antibody incubation, wash 3x with 1x PBS.</li>
	<li>Remove the chambers, either by simply removing them by hand or using the tool that comes with the slides. Follow the directions printed on the chamber slide packaging for removal.</li>
	<li>Add a drop of 4&#8217;,6-diamidino-2-phenylindole (DAPI) mounting serum and place a coverslip on the top. Limit the number of bubbles and make sure all the cells are covered. Optional step: use nail polish around the edges to&#160;seal coverslip.</li>
	<li>Lay the slide flat in a slide box to dry and place at 4 &#176;C until imaging.</li>
	<li>Image on a microscope equipped with correct fluorescence based on secondary antibody conjugation to ensure clean image.</li>
</ol>

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The authors have no conflicts of interest to declare.

There is a considerable amount of interest in studying fallopian tube epithelium as fallopian tubes play a significant role in reproduction and are the site of origin for most HGSOC. To that end, many investigators have described protocols to isolate fallopian tube cells in both human and mouse models10,11,12,15,16,17,18,19,20,21. The method we describe to extract and enrich for fallopian tube epithelial cells adds to existing fallopian tube cell isolation protocols. Although overlaps exist within these protocols, there are two general types of methods reported in mice and humans. The first involves mincing and enzymatically digesting the whole fallopian tube that gives a total cell suspension10,12,15,16,17. The second involves sloughing with agitation or scraping, which results in sheets of tissue11,18,19,20,21. Both methods allow epithelial cells to be analyzed via downstream experiments such as flow cytometry, sequencing, and in-vitro culture. A major advantage of our method is that the protocol yields a population of fallopian tube cells that are already enriched for epithelial cells through enzymatic digestion and mechanical pushing steps. This population enriched for epithelia can be further digested resulting in a single-cell suspension that can be used for many applications such as flow cytometry, 2D culture, immunocytochemistry, and single-cell RNA sequencing.
Key steps we found to impact cell yield include the duration for which the fallopian tube fragments incubate in 1% trypsin/HBSS and DMEM/DNase. Overincubation in either solution will degrade the cells and significantly decrease the viability of the cells. However, insufficient incubation time will inhibit the researcher&#39;s ability to push out many epithelial cells during protocol step 2.6 as they will continue to adhere to each other. It is also important to use a 1% trypsin solution, as both lower and higher concentrations of trypsin solutions reduced the yield of viable fallopian tube epithelial cells. By combining chemical and mechanical isolation and a digestion step (protocol step 3.1), we can exponentially shorten the period it takes to go from tissue to single-cell suspension. This ensures good viability and time to perform downstream analysis on the same day.
In protocol step 1.4, it is critical to ensure that the pieces of fallopian tube cut are 3-5 mm thick. If the pieces are too large, it will be difficult to perform protocol step 2.7 and ultimately decrease the cell yield as a longer incubation time in DMEM/DNase will be necessary.
Although the cell suspension is enriched for epithelial cells, stromal cell contamination is inevitable. If the preparation needs to be purely epithelial cells, sorting using flow cytometry and the markers we described can be performed to isolate epithelial cells and deplete stromal cells.
Postmenopausal fallopian tubes were used in this study. However, this method has been successfully utilized in our lab on premenopausal fallopian tubes. The main difference between pre- and postmenopausal fallopian tubes is the composition of ciliated and secretory epithelial cells1. This protocol works for all reproductive stages.
This efficient protocol will facilitate investigating cell types of&#160;the fallopian tube epithelium, including delineating cellular lineages, their dynamic changes during reproductive cycles as well as after menopause, and their role in initiating high-grade serous ovarian cancer.

The fallopian tubes are constructed of multiple parts. From the ovary to the uterus, the fallopian tube is composed of the fimbriae, ampulla, isthmus, and the intramural portion. The fimbriae extend from the end of the fallopian tube where they seize the ovum released by the ovary. The ovum then travels through the ampulla, where it is most likely to be fertilized, to the isthmus and finally is transferred to the uterus through the intramural portion1. The innermost mucosa of the fallopian tube that facilitates ovum transportation is made up of a layer of luminal epithelium, including ciliated and secretory cells. Ciliated cells tend to be more concentrated at the fimbriae2. They play an integral role in physically moving the ovum through the fallopian tube from the ovary to the uterus. Their appendages allow the ciliated cells to not only move the ovum along but also to clear out genotoxic stress following ovulation3.
Secretory fallopian tube epithelial cells secrete a fluid that aids in nutrition and gamete assembly4. The proportion of ciliated and secretory cells along the fallopian tube epithelium differs in the post-menopausal state with a decrease in ciliated cells as the fallopian tube no longer serves a critical function in transportation5. Furthermore, in the absence of estrogen, the fallopian tubes are thought to become vestigial1,6. This loss of ciliated cells in the fallopian tube is posited to heighten the risk of developing serous carcinomas7. Additionally, fallopian tube secretory epithelial cells are thought to give rise to serous tubal intraepithelial carcinoma (STIC) lesions, a well-known precursor to the most aggressive subtype of tubo ovarian cancers, high grade serous carcinoma7,8.
The purpose of this protocol is to isolate epithelial cells from human fallopian tubes and dissociate them into single-cell suspensions. This protocol yields an enriched single-cell epithelial population that can be used for many analyses. As shown in this manuscript, we have performed flow cytometry analysis and plated cells in 2D after isolation. Flow cytometry analysis demonstrates the presence of single cells, which are mostly viable and epithelial in nature. In these analyses, we included four markers, e506 for viability, EpCAM for epithelial cells, CD45 for immune cells, and CD10 for stromal cells. Dead cells were excluded using the e506 viability marker, and immune cells were gated out using CD45. It is possible for the suspension to have an immune cell population; however, to achieve a relatively pure population of epithelial cells, CD45-positive cells can be depleted using a CD45 depletion kit. Further, when plated in culture, the CD45-positive cells often do not proliferate. Cells isolated via this method and grown in 2D show adherent cobblestone-like epithelial populations. This method can be used to generate cellular preparations, which can be developed into single-cell RNA libraries.
Research to define the cellular lineage of fallopian tube epithelia, alterations in these lineages during different phases of reproductive life, and inciting events that lead to malignant transformation and genesis has become more prominent within the last four years6,9,10,11,12.This protocol will significantly benefit research in this arena by providing an efficient way to isolate fallopian tube epithelia and process them into single cells.

<table><tbody><tr><td>1% Trypsin&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>J63993.09</td><td></td></tr><tr><td>100 &amp;micro;m Cell Strainer</td><td>Corning</td><td>431752</td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710-S</td><td></td></tr><tr><td>5 mL round bottom tubes</td><td>Corning</td><td>352008</td><td></td></tr><tr><td>60 mm cell culture plate</td><td>Corning&amp;nbsp;</td><td>CLS430589-500EA</td><td></td></tr><tr><td>6-well Cell Culture Plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Anti-CD10</td><td>BioLegend</td><td>312212</td><td></td></tr><tr><td>Anti-CD326 (Ep-CAM)</td><td>BioLegend</td><td>324218</td><td></td></tr><tr><td>Anti-CD45&amp;nbsp;</td><td>BioLegend</td><td>304026</td><td></td></tr><tr><td>Anti-EpCAM</td><td>Abcam</td><td>ab223582</td><td></td></tr><tr><td>Anti-IgG2a PerCP&amp;nbsp;</td><td>BioLegend</td><td>400250</td><td></td></tr><tr><td>Anti-PAX8</td><td>Sigma-Aldrich</td><td>363M-15</td><td></td></tr><tr><td>Anti-Vimentin</td><td>Agilent Technologies</td><td>M072501-2</td><td></td></tr><tr><td>Chamber Slide System</td><td>Thermo Scientific&amp;nbsp;</td><td>154917PK</td><td></td></tr><tr><td>Collagenase&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>17100017</td><td></td></tr><tr><td>DMEM</td><td>Thermo Scientific&amp;nbsp;</td><td>10569-010</td><td></td></tr><tr><td>DNase I</td><td>Sigma</td><td>10104159001</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>3690</td><td></td></tr><tr><td>eFluor 506</td><td>Invitrogen</td><td>65-0866-14</td><td></td></tr><tr><td>FBS</td><td>Sigma</td><td>F2442</td><td></td></tr><tr><td>Fine point forceps&amp;nbsp;</td><td>VWR</td><td>102091-526</td><td>Any finepoint forceps of your choice will work</td></tr><tr><td>Fixable Viability Dye eFluor 506</td><td>Invitrogen</td><td>65-0866-14</td><td></td></tr><tr><td>FlowJo software version 9&amp;nbsp;</td><td>BD Biosciences</td><td></td><td>Data analysis software</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>HBSS</td><td>Thermo Scientific&amp;nbsp;</td><td>14175-095</td><td></td></tr><tr><td>MACSQuant Analyzer 10 flow cytometer&amp;nbsp;</td><td>Miltenyi Biotec</td><td></td><td></td></tr><tr><td>MACSQuant Calibration Beads</td><td>Miltenyi Biotec</td><td>130-093-607</td><td></td></tr><tr><td>Mammocult&amp;nbsp;</td><td>Stemcell Technologies</td><td>5620</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Fisher Scientific</td><td>PI31873</td><td></td></tr><tr><td>PBS&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>14190-144</td><td></td></tr><tr><td>Penicillin-Streptomyocin&amp;nbsp;</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>PerCP Conjugated CD45</td><td>BioLegend</td><td>304026</td><td></td></tr><tr><td>Red Blood Cell lysis buffer&amp;nbsp;</td><td>Tonbo Biosciences</td><td>TNB-4300-L100&amp;nbsp;</td><td></td></tr><tr><td>Triton X-100</td><td>Thermo Scientific&amp;nbsp;</td><td>BP151-100</td><td></td></tr><tr><td>Vannas-Tubingen Spring Scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>VECTASHIELD with DAPI</td><td>Fisher Scientific</td><td>NC9524612</td><td></td></tr></tbody></table>

an efficient method for extracting human fallopian tube epithelia for single-cell analyses

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if fertilization is successful, the embryo. The epithelial cells that line the inner surface of fallopian tubes are integral to both normal and abnormal fallopian tube processes, including disease initiation. After menopause, fallopian tubes cease to have a significant role in the body and the intraepithelial cell makeup changes. We describe a method in which these epithelial cells can be isolated from fresh fallopian tubes with minimal stromal cell contamination into a single-cell suspension. These cells can be grown in culture or used for further analysis such as flow cytometry and single-cell RNA sequencing. This isolation protocol can be achieved in 4-6 h and yields viable cells that can be used for immediate downstream analysis. This efficient protocol facilitates the isolation of fallopian tube epithelial cells with an enriched epithelial population.

We have included seven fallopian tube collections where we have isolated an enriched epithelial cell population (Figure 2A and Supplemental Figure S1A). To assess the viability and epithelial cell enrichment of this single-cell suspension method, flow cytometry was performed. To measure the cell viability, cells were stained with the viability marker, e506. This also allowed for gating out all debris and dead cells when analyzed.
To determine the composition of cells isolated via this method, samples were stained with an epithelial cell marker (EpCAM), a stromal cell marker (CD10), and an immune cell marker (CD45). As seen in Figure 2B, we isolated a viable and enriched epithelial cell population from fallopian tube tissue. Sample viability was at an average of 82%. For all samples, after gating out CD45-positive cells, we observed an enriched epithelial cell population with EpCAM-positive cells averaging 80% of the sample. There was some stromal cell contamination but only 7.8% on average. Figure 2C shows isolated cells in 2D&#160;4-6 days after plating. It is apparent that epithelial cells were isolated as they formed consistent adherent cobblestone-looking clusters. In Supplemental Figure S1B,&#160;cells were plated after isolation and cultured for 2-6 days. Immunocytochemistry was used to characterize the cells growing in culture. Most cells in the culture stained positive for EpCAM and the secretory cell marker, PAX8. Only a few cells were identified as vimentin-positive. Ciliated cells were seen in culture as captured by video, shown in Supplemental Video S1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67753/67753fig1.jpg" /> 
Figure 1: Experimental schema. (A) Fallopian tubes are acquired. (B) Excess fat and connective tissue are removed. (C) Tubes are cut into ~3-5 mm pieces. (D) The pieces are washed in PBS, then incubated in EDTA 2x for 5 min, and incubated in 1% trypsin for 40 min at 4 &#730;C. (E) Using two pairs of forceps, one to hold and one to push, expel epithelial cells from the fallopian tube pieces. (F) Transfer the cells to a 15 mL tube, spin, and count. (G) Resuspend in DMEM, collagenase, and DNase. Digest for 30-45 min. (H) Strain with a 100 &#181;m strainer. (I) Harvest by centrifugation and count. <a href="https://www.jove.com/files/ftp_upload/67753/67753fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67753/67753fig2.jpg" /> 
Figure 2: Characterization of isolated epithelial cells. (A) Table of patient clinical information and flow cytometry results. (B)&#160;Flow cytometry was performed after isolation to show that an enriched population of epithelial cells (EpCAM-positive) had been isolated. Isotype negative control is indicated in black, and the experimental result is indicated in pink. (C) Cells were plated after isolation. Photos were taken 4-6 days after plating. Scale bars = 100 &#181;m. Abbreviations: AUB = Abnormal uterine bleeding; EIN = Endometrial intraepithelial neoplasia. <a href="https://www.jove.com/files/ftp_upload/67753/67753fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure S1: Additional characterization of isolated epithelial cells in culture. (A) Table of patient clinical information. (B) Isolated cells were cultured and stained for EpCAM, a secretory cell marker (PAX8), and a stromal cell marker (Vimentin) 2-6 days after plating to characterize cell type in culture. Scale bars = 50 &#181;m. Abbreviations: EIN: Endometrial intraepithelial neoplasia; DAPI = 4&#39;,6-diamidino-2-phenylindole.&#160;<a href="https://www.jove.com/files/ftp_upload/67753/67753_Supplemental Figure 1 (2).pdf" target="_blank">Please click here to download this Figure.</a>
Supplemental Video S1: Video of beating ciliated cells were taken at 30x magnification to demonstrate ciliated cells in culture. <a href="https://www.jove.com/files/ftp_upload/67753/Supplemental Video 1 FT4.mp4" target="_blank">Please click here to download this Video.</a>

This protocol describes a one-day method for the isolation of human fallopian tube epithelial cells. Isolated epithelial cells can be plated in 2-dimensional (2D) culture or dissociated into single-cell suspensions and utilized in downstream experiments, including flow cytometry and single-cell RNA sequencing.

An Efficient Method for Extracting Human Fallopian Tube Epithelia for Single-cell Analyses

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if ...

an-efficient-method-for-extracting-human-fallopian-tube-epithelia-for

Research

JoVE Journal

Cancer Research

499 Views.  University of California, Los Angeles. Dit protocol beschrijft een eendaagse methode voor de isolatie van menselijke eileiderepitheelcellen. Geïsoleerde epitheelcellen kunnen worden geplateerd in 2-dimensionale (2D) cultuur of worden gedissocieerd in eencellige suspensies en worden gebruikt in stroomafwaartse experimenten, waaronder flowcytometrie en eencellige RNA-sequencing. 

Video: Een efficiënte methode voor het extraheren van epitelie van de menselijke eileider voor analyses van één cel

Intra-iliac Artery Injection for Efficient and Selective Modeling of Microscopic Bone Metastasis

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely used intra-cardiac and intra-tibial injections, IIA injection brings several advantages. First, it can deliver a large quantity of cancer cells specifically to hind limb bones, thereby providing spatiotemporally synchronized early-stage colonization events and allowing robust quantification and swift detection of disseminated tumor cells. Second, it injects cancer cells into the circulation without damaging the local tissues, thereby avoiding inflammatory and wound-healing processes that confound the bone colonization process. Third, IIA injection causes very little metastatic growth in non-bone organs, thereby preventing animals from succumbing to other vital metastases, and allowing continuous monitoring of indolent bone lesions. These advantages are especially useful for the inspection of progression from single cancer cells to multi-cell micrometastases, which has largely been elusive in the past. When combined with cutting-edge approaches of biological imaging and bone histology, IIA injection can be applied to various research purposes related to bone metastases.

This manuscript provides the detailed procedure of intra-iliac artery (IIA) injection, a technique to deliver cancer cells specifically to hind limb tissues including bones to establish experimental bone metastases. Although initially established with breast tumor models, this protocol can be easily extended to other cancer types.

Intra-iliac artery (IIA) injection is an efficient approach to introduce metastatic lesions of various cancer cells in animals. Compared to the widely ...

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue organization. Drosophila melanogaster has emerged as a popular experimental model system in cancer biology to study the genetic and cellular mechanisms of tumorigenesis. In particular, genetic tools for Drosophila imaginal discs (developing epithelia in larvae) enable the creation of transformed pro-tumor cells within a normal epithelial tissue, a situation similar to the initial stages of human cancer. A recent study of tumorigenesis in Drosophila wing imaginal discs, however, showed that tumor initiation depends on the tissue-intrinsic cytoarchitecture and the local microenvironment, suggesting that it is important to consider the region-specific susceptibility to tumorigenic stimuli in evaluating tumor phenotypes in imaginal discs. To facilitate phenotypic analysis of tumor progression in imaginal discs, here we describe a protocol for genetic experiments using the GAL4-UAS system to induce neoplastic tumors in wing imaginal discs. We further introduce a diagnosis method to classify the phenotypes of clonal lesions induced in imaginal epithelia, as a clear classification method to discriminate various stages of tumor progression (such as hyperplasia, dysplasia, or neoplasia) had not been described before. These methods might be broadly applicable to the clonal analysis of tumor phenotypes in various organs in Drosophila.

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Mosaic clone analysis in Drosophila imaginal disc epithelia is a powerful model system to study the genetic and cellular mechanisms of tumorigenesis. Here we describe a protocol to induce tumors in Drosophila wing imaginal discs using the GAL4-UAS system, and introduce a diagnosis method to classify the tumor phenotypes.

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and protein functions, and cell signaling aberrations may therefore serve as biomarkers to indicate personalized treatment options. As opposed to DNA and RNA analyses, changes in protein activity can more efficiently evaluate the mechanisms underlying drug sensitivity and resistance. Phospho flow cytometry is a powerful technique that measures protein phosphorylation events at the cellular level, an important feature that distinguishes this method from other antibody-based approaches. The method allows for simultaneous analysis of multiple signaling proteins. In combination with fluorescent cell barcoding, larger medium- to high-throughput data-sets can be acquired by standard cytometer hardware in short time. Phospho flow cytometry has applications both in studies of basic biology and in clinical research, including signaling analysis, biomarker discovery and assessment of pharmacodynamics. Here, a detailed experimental protocol is provided for phospho flow analysis of purified peripheral blood mononuclear cells, using chronic lymphocytic leukemia cells as an example.

Here, a protocol for medium- to high-throughput analysis of protein phosphorylation events at the cellular level is presented. Phospho flow cytometry is a powerful approach to characterize signaling aberrations, identify and validate biomarkers, and assess pharmacodynamics.

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

An Automated Microscopic Scoring Method for the &#947;-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects induced by exposure to ionizing radiation to make an individual dose assessment. This is pertinent in the framework of radiation emergencies, where health assessments and planning of clinical treatment for exposed victims are critical. Since DNA double strand breaks (DSB) are considered to be the most lethal form of radiation-induced DNA damage, this protocol presents a method to detect DNA DSB in blood samples. The methodology is based on the detection of a fluorescent labelled phosphorylated DNA repair protein, namely, &#947;-H2AX. The use of an automated microscopy platform to score the number of &#947;-H2AX foci per cell allows a standardized analysis with a significant decrease in the turn-around time. Therefore, the &#947;-H2AX assay has the potential to be one of the fastest and sensitive assays for biological dosimetry. In this protocol, whole blood samples from healthy adult volunteers will be processed and irradiated in vitro in order to illustrate the usage of the automated and sensitive &#947;-H2AX foci assay for biodosimetry applications. An automated slide scanning system and analysis platform with an integrated fluorescence microscope is used, which allows the fast, automatic scoring of DNA DSB with a reduced degree of bias.

This protocol presents the slide preparation and automated scoring of the &#947;-H2AX foci assay on peripheral blood lymphocytes. To illustrate the method and sensitivity of the assay, isolated lymphocytes were irradiated in vitro. This automated method of DNA DSB detection is useful for fast and high-throughput biological dosimetry applications.

Ionizing radiation is a potent inducer of DNA damage and a well-documented carcinogen. Biological dosimetry comprises the detection of biological effects ...

Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion

The lack of suitable translational research models reflecting primary disease to explore tumorigenesis and therapeutic strategies is a major obstacle in esophageal adenocarcinoma (EAC). Patient-derived organoids (PDOs) have recently emerged as a remarkable preclinical model in a variety of cancers. However, there are still limited protocols available for developing EAC PDOs. Once the PDOs are established, the propagation and cryopreservation are essential for further downstream analyses. Here, two different methods have been standardized for EAC PDOs subculture and cryopreservation, i.e., with and without single cell digestion. Both methods can reliably obtain appropriate cell viability and are applicable for a diverse experimental setup. The current study demonstrated that subculturing EAC PDOs with single cell digestion is suitable for most experiments requiring cell number control, uniform density, and a hollow structure that facilitates size tracking. However, the single cell-based method shows slower growth in culture as well as after re-cultivation from frozen stocks. Besides, subculturing with single cell digestion is characterized by forming hollow structures with a hollow core. In contrast, processing EAC PDOs without single cell digestion is favorable for cryopreservation, expansion, and histological characterization. In this protocol, the advantages and disadvantages of subculturing and cryopreservation of EAC PDOs with and without single cell digestion are described to enable researchers to choose an appropriate method to process and investigate their organoids.

This protocol describes the methods of subculture and cryopreservation of esophageal adenocarcinoma organoids with and without single cell digestion to enable researchers to choose appropriate strategies based on their experimental design.

The lack of suitable translational research models reflecting primary disease to explore tumorigenesis and therapeutic strategies is a major obstacle in ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

An Efficient Method for Extracting Human Fallopian Tube Epithelia for Single-cell Analyses

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