Name: a single cell dissociation approach for molecular analysis of urinary bladder in the mouse following spinal cord injury
Uploaded: 2020-06-17T15:00:00.000+00:00
Duration: 9 min 50 s
Description: Watch this Scientific Journal Video about A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury at JoVE.com

This work was supported by grants from the National Institutes of Health (R01 DK077195 to R.M.A, R01 DK104641 to R.M.A and D.R.B). We acknowledge valuable input from Dr. Stuart Orkin in the Division of Hematology/Oncology, Boston Children&#8217;s Hospital, Department of Pediatrics, Harvard Medical School and the Dana-Farber Cancer Institute. We also acknowledge support from Kyle Costa in post-operative care of mice, Mary Taglienti and Dr. Habiballah Shojaeisaadi (Dr. Yang Shi Laboratory, Dept. of Pediatrics, Division of Newborn Medicine, Dept. of Pediatrics, Division of Newborn Medicine, Boston Children&#8217;s Hospital, Harvard Medical School) for technical assistance and helpful discussions.

<ol>
	<li>Grange, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+and+functional+analysis+of+human+tumor+immune+infiltration+after+mechanical+and+enzymatic+disaggregation.">Phenotypic characterization and functional analysis of human tumor immune infiltration after mechanical and enzymatic disaggregation.</a> Journal of Immunological Methods. 372 (1-2), 119-126 (2011).</li><li>van den Brink, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+sequencing+reveals+dissociation-induced+gene+expression+in+tissue+subpopulations.">Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations.</a> Nature Methods. 14 (10), 935-936 (2017).</li><li>Seth, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+discrete+modes+of+spinal+cord+injury+on+bladder+muscle+contractility.">The impact of discrete modes of spinal cord injury on bladder muscle contractility.</a> BMC Urology. 13, 24 (2013).</li><li>Doyle, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inosine+attenuates+spontaneous+activity+in+the+rat+neurogenic+bladder+through+an+A2B+pathway.">Inosine attenuates spontaneous activity in the rat neurogenic bladder through an A2B pathway.</a> Scientific Reports. 7, 44416 (2017).</li><li>Gheinani, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+miRNA-regulated+networks,+hubs+of+signaling,+and+biomarkers+in+obstruction-induced+bladder+dysfunction.">Characterization of miRNA-regulated networks, hubs of signaling, and biomarkers in obstruction-induced bladder dysfunction.</a> JCI Insight. 2 (2), 89560 (2017).</li><li>Gheinani, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concordant+miRNA+and+mRNA+expression+profiles+in+humans+and+mice+with+bladder+outlet+obstruction.">Concordant miRNA and mRNA expression profiles in humans and mice with bladder outlet obstruction.</a> American Journal of Clinical and Experimental Urology. 6 (6), 219-233 (2018).</li><li>Krishna, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+contusion+model+of+severe+spinal+cord+injury+in+rats.">A contusion model of severe spinal cord injury in rats.</a> Journal of Visualized Experiments. (78), e50111 (2013).</li><li>The Tabula Muris Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomics+of+20+mouse+organs+creates+a+Tabula+Muris.">Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris.</a> Nature. 562 (7727), 367-372 (2018).</li><li>Gray, C. J., Boukouvalas, J., Szawelski, R. J., Wharton, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzyloxycarbonylphenylalanylcitrulline+p-nitroanilide+as+a+substrate+for+papain+and+other+plant+cysteine+proteinases.">Benzyloxycarbonylphenylalanylcitrulline p-nitroanilide as a substrate for papain and other plant cysteine proteinases.</a> Biochemical Journal. 219 (1), 325-328 (1984).</li><li>Feodorova, Y., Koch, M., Bultman, S., Michalakis, S., Solovei, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quick+and+reliable+method+for+retina+dissociation+and+separation+of+rod+photoreceptor+perikarya+from+adult+mice.">Quick and reliable method for retina dissociation and separation of rod photoreceptor perikarya from adult mice.</a> MethodsX. 2, 39-46 (2015).</li><li>Aitken, K. J., Bagli, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bladder+extracellular+matrix.+Part+I:+architecture,+development+and+disease.">The bladder extracellular matrix. Part I: architecture, development and disease.</a> Nature Reviews Urology. 6 (11), 596-611 (2009).</li><li>Aitken, K. J., Bagli, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bladder+extracellular+matrix.+Part+II:+regenerative+applications.">The bladder extracellular matrix. Part II: regenerative applications.</a> Nature Reviews Urology. 6 (11), 612-621 (2009).</li><li>The ENCODE Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Yue, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+encyclopedia+of+DNA+elements+in+the+mouse+genome.">A comparative encyclopedia of DNA elements in the mouse genome.</a> Nature. 515 (7527), 355-364 (2014).</li><li>Wu, Y. E., Pan, L., Zuo, Y., Li, X., Hong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+Activated+Cell+Populations+Using+Single-Cell+RNA-Seq.">Detecting Activated Cell Populations Using Single-Cell RNA-Seq.</a> Neuron. 96 (2), 313-329 (2017).</li></ol>

No conflicts of interest declared.

The mouse spinal cord injury model described here provides a reproducible method to create a functional bladder outlet obstruction due to loss of coordination between bladder contraction and external urethral sphincter relaxation. This in turn evokes profound remodeling of the bladder wall as early as 2 weeks after injury characterized by expansion of urothelial and smooth muscle compartments. Critical steps in implementation of the SCI model in rodents include (i) rigorous attention to manual bladder expression during the period of spinal shock that ensues for 10&#8210;14 days after injury; (ii) nutritional enrichment to minimize weight loss; and (iii) mitigation of the potential for urine scalding particularly for experiments that extend beyond the return of reflex voiding. Limitations of the model include the potential for urethral occlusion in mice from blood clots during the period of transient hematuria, and additionally in male mice from semen coagulum following retrograde ejaculation following surgery.
The tissue dissociation approach described here illustrates the importance of considering structural changes in tissues that arise from the experimental insult, in this case significant tissue remodeling following SCI that may influence downstream analyses. With the increase in single cell analyses it is critical to ensure that differences observed in gene expression are not simply a result of dissociation-induced perturbations, but are truly representative of underlying biological changes relevant to the disease model. The use of publicly available expression data allowed us to modify the formulation of digestion buffers to ensure effective digestion of extracellular matrix while maximizing viability. Additional modifications that could be considered in future applications include the addition of actinomycin D, to halt transcription of immediate early genes that are sensitive to the dissociation protocol15.
Pipetting technique is crucial when dissociating tissue or transferring cells that are already in suspension. To reduce physical damage to cells from shearing forces, it is important to pipette gently and slowly during cell resuspension. It is generally recommended to use wide-bore pipette tips. If using standard tips, it is particularly important to pipette cell suspensions gently to avoid shear forces that would otherwise damage cells. Using cell strainers is unavoidable in this protocol, however, the cell concentration can decline by 20% or more, accompanied by a volume loss of 100 &#181;L or more. We recommend that cell concentration be determined after straining to ensure a precise cell count.
In flow cytometry, FMO controls provide a measure of background due to bleed-through of signal from overlapping emission peaks. They are not a measure of nonspecific antibody binding, or background staining that may be present when an antibody is included in that channel. To account for the nonspecific antibody binding, one has to include appropriate isotype controls; for the background staining, one needs to include negative controls. Taken together, these controls ensure accurate measurement of cell populations.

Perturbations of normal urinary bladder function can lead to decreased quality of life for many individuals. In order to gain a better understanding of how injury or disease derails normal bladder function, it is important to probe the normal biological state of cells within the bladder and how they change under experimental perturbation. To date, however, the specific cell populations that reside within the urinary bladder, and how they change with injury, have been incompletely characterized.
Single cell profiling methods such as flow cytometry or single cell RNA sequencing (scRNA-seq) have the potential to shed light on specific cell types within the bladder. However, for these approaches to be informative tissue must be digested in a manner that does not affect viability, gene expression, and representative cell population percentages of the harvested tissue. Protocols that employ enzymatic disaggregation can impact surface marker expression through indiscriminate protease activity1, thereby impacting cell identification by flow cytometry, whereas the dissociation process itself can lead to the induction of immediate early genes, as described recently by Van den Brink and colleagues2. The authors showed that although the dissociation-affected subpopulation was small, it could trigger a strong contaminating signal in bulk expression studies due to the high expression levels of immediate early genes. In addition, the duration of the dissociation protocol affected detected bulk expression levels of genes shown to be unique to some subpopulations. Thus, single cell datasets generated without accounting for the impact of the dissociation protocol may yield gene expression changes arising from the dissociation method, as opposed to underlying biology. These observations suggest that published single cell transcriptomics data should be interpreted with caution, and that results should be validated by independent methods.
Although, harsh and lengthy dissociation methods may alter gene expression in cells2; effective isolation of cells is essential to obtain accurate representation of the cell types present. Since the bladder is a complex organ comprising multiple cell types, some populations such as urothelial or stromal cells may be relatively under-represented whereas other cell types such as fibroblasts exist within extracellular matrix and can be challenging to isolate. Dissociation becomes even more challenging if the bladder has undergone significant remodeling and fibrosis such as that observed in spinal cord injury3,4 or bladder outlet obstruction5,6.
Here, we describe an optimized tissue dissociation method for downstream single cell analysis in the spinal cord injured mouse bladder. Using flow cytometry, we compared four enzymatic digestion protocols for their ability to yield a single cell suspension, support cell viability and maintain the correct proportion of cell populations. Based on this analysis, we conclude that minimizing cell death, cellular aggregates, non-cellular nucleic acids and potential inhibitors of downstream analysis are critical to achieving high quality data.

<table><tbody><tr><td>2.5 X Magnifying Loupes</td><td></td><td></td><td></td></tr><tr><td>7-0 Vicryl suture, 6.5mm needle 3/8 circle</td><td>ETHICON</td><td>J546</td><td></td></tr><tr><td>70 &amp;mu;m Cell Strainer</td><td>Thermofisher</td><td>22363548</td><td></td></tr><tr><td>Accutase in BPBS, 0.5mM EDTA</td><td>Millipore</td><td>SCR005</td><td></td></tr><tr><td>Aerosol Filter Wide Orifice Pipettor Tips (1000 &amp;micro;L)</td><td>VWR</td><td>89049-168</td><td></td></tr><tr><td>Aerosol Filter Wide Orifice Pipettor Tips (1000 &amp;micro;L)</td><td>VWR</td><td>89049-168</td><td></td></tr><tr><td>APC anti-mouse CD326 (Ep-CAM), rat monoclonal, IgG2a, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>118213</td><td></td></tr><tr><td>BB515 Rat Anti-Mouse CD45, rat monoclonal, IgG2b, &amp;kappa;, Clone 30-F11</td><td>BD Biosciences</td><td>564590</td><td></td></tr><tr><td>BONN Micro Dissecting Forceps, Straight, 1x2 teeth, 3.75&quot; length, 0.3mm tip width, 0.12mm teeth</td><td>ROBOZ Surgical Instrument Company, Inc.</td><td>RS-5172</td><td>ROBOZ Surgical Instrument Company, Inc., Gaithersburg MD</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A9647-100G</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>2115-250ML</td><td></td></tr><tr><td>CASTROVIEJO Micro Suturing Needle Holder, Straight with lock, 5.75&quot; length</td><td>ROBOZ Surgical Instrument Company, Inc.</td><td>RS-6412</td><td>ROBOZ Surgical Instrument Company, Inc., Gaithersburg MD</td></tr><tr><td>Cell Counting Kit, 30 dual-chambered slides, 60 counts, with trypan blue</td><td>Biorad</td><td>1450003</td><td></td></tr><tr><td>Cell Staining Buffer</td><td>BioLegend</td><td>420201</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum</td><td>Sigma</td><td>C0130-1G</td><td></td></tr><tr><td>Collagenase Type I</td><td>Worthington Biochemical Corporation</td><td>LS004196</td><td></td></tr><tr><td>Collagenase Type III</td><td>Worthington Biochemical Corporation</td><td>LS004182</td><td></td></tr><tr><td>Collagenase, Type 6</td><td>Worthington Biochemical Corporation</td><td>LS005319</td><td></td></tr><tr><td>Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 &amp;amp; Propidium Iodide (PI)</td><td>Thermofisher</td><td>V13241</td><td></td></tr><tr><td>Dispase II</td><td>Sigma</td><td>D4693-1G</td><td></td></tr><tr><td>DNase</td><td>Sigma</td><td>DN25-1G</td><td></td></tr><tr><td>Enrofloxacin (Baytril)</td><td>Bayer Health Care LLC,</td><td>NADA # 140-913 Approved by FDA. Lot No.: AH01CGP</td><td>2.27% Injectable Solution</td></tr><tr><td>Falcon 15 ml conical centrifuge tubes</td><td>Fisher Scientific</td><td>352096</td><td></td></tr><tr><td>Falcon 50 ml conical centrifuge tubes</td><td>Fisher Scientific</td><td>352070</td><td></td></tr><tr><td>FITC anti-mouse Ly-6A/E (Sca-1) Antibody, rat monoclonal, IgG2a, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>122505</td><td></td></tr><tr><td>Hyaluronidase from sheep testes, Type II</td><td>Sigma</td><td>H2126</td><td></td></tr><tr><td>MACS SmartStrainers (100 &amp;micro;m)</td><td>Miltenyi Biotec, Inc.</td><td>130-110-917</td><td></td></tr><tr><td>McPHERSON-VANNAS, Micro Dissecting Spring Scissors, Straight, 4&quot; length, 0.15mm tip width</td><td>ROBOZ Surgical Instrument Company, Inc.</td><td>RS-5630</td><td>ROBOZ Surgical Instrument Company, Inc., Gaithersburg MD</td></tr><tr><td>Meloxicam</td><td>Patterson Veterinary</td><td>07-891-7959</td><td></td></tr><tr><td>Papain</td><td>Worthington Biochemical Corporation</td><td>LS003119</td><td></td></tr><tr><td>PE/Cy5 anti-mouse CD19 Antibody, rat monoclonal, IgG2a, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>115509</td><td>Dump Channel</td></tr><tr><td>PE/Cy5 anti-mouse CD3&amp;epsilon; Antibody, Armenian hamster monoclonal, IgG, affinity purified</td><td>BioLegend</td><td>100309</td><td>Dump Channel</td></tr><tr><td>PE/Cy5 anti-mouse CD4 Antibody, rat monoclonal, IgG2b, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>100409</td><td>Dump Channel</td></tr><tr><td>PE/Cy5 anti-mouse CD8a Antibody, rat monoclonal, IgG2a, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>100709</td><td>Dump Channel</td></tr><tr><td>PE/Cy5 anti-mouse NK-1.1 Antibody, mouse monoclonal, IgG2a, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>108715</td><td>Dump Channel</td></tr><tr><td>PE/Cy5 anti-mouse TER-119/Erythroid Cells Antibody, IgG2b, &amp;kappa;, affinity purified</td><td>BioLegend</td><td>116209</td><td>Dump Channel</td></tr><tr><td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block), rat monoclonal, IgG2b, &amp;kappa;, Clone 2.4G2</td><td>BD Biosciences</td><td>553141</td><td></td></tr><tr><td>RBC Lysis Buffer (10X)</td><td>BioLegend</td><td>420301</td><td></td></tr><tr><td>Red Blood Cell Lysis Buffer 1x</td><td>Biolegend</td><td>420201</td><td></td></tr><tr><td>Screw-Cap microcentrifuge tubes, 1.5 ml</td><td>VWR</td><td>89004-290</td><td></td></tr><tr><td>TC20 Automated Cell Counter</td><td>Biorad</td><td>1450102</td><td></td></tr><tr><td>Triple antibiotic ointment (neomycin/polymyxin B/ bacitracin)</td><td>Patterson Veterinary</td><td>07-893-7216</td><td>skin protectant</td></tr><tr><td>TrypLE Select Enzyme (10X), no phenol red</td><td>Thermofisher</td><td>A1217701</td><td></td></tr><tr><td>Vetropolycin eye ointment</td><td>Dechra Veterinary Products</td><td>NADA # 065-016. Approved by FDA.</td><td>protect eyes during anesthesia</td></tr></tbody></table>

a single cell dissociation approach for molecular analysis of urinary bladder in the mouse following spinal cord injury

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and subsequent bladder wall remodeling. To facilitate assessment of the cellular composition of the bladder wall in non-injured control and spinal cord injured mice, we developed an optimized dissociation protocol that supports high cell viability and enables the detection of discrete subpopulations by flow cytometry.
Spinal cord injury is created by complete transection of the thoracic spinal cord. At the time of tissue harvest, the animal is perfused with phosphate-buffered saline under deep anesthesia and bladders are harvested into Tyrode&#8217;s buffer. Tissues are minced prior to incubation in digestion buffer that has been optimized based on the collagen content of mouse bladder as determined by interrogation of publicly available gene expression databases. Following generation of a single cell suspension, material is analyzed by flow cytometry for assessment of cell viability, cell number and specific subpopulations. We demonstrate that the method yields cell populations with greater than 90% viability, and robust representation of cells of mesenchymal and epithelial origin. This method will enable accurate downstream analysis of discrete cell types in mouse bladder and potentially other organs.

Surgical procedure 
The success of thoracic spinal cord transection is determined by assessment of a number of parameters, the most obvious of which is hindlimb paralysis. The animal moves only using its forelimbs, dragging its hindlimbs. Otherwise activity levels, including feeding, grooming and alertness are typically normal. In addition, animals lose volitional bladder control resulting in the need for manual bladder expression by the investigator every 12 h until reflex voiding returns at 10 to 14 days after injury. Following euthanasia, additional signs of the success of the injury relate primarily to the increase in bladder-to-body weight ratio, indicative of tissue remodeling. Histological analysis reveals hyperplasia within both the urothelial and smooth muscle compartments3.
Preparation of single cell suspension 
Using publicly available expression data, the enrichment of bladder tissue for extracellular matrix proteins was determined (Figure 2) and used to inform the formulation of the digestion mix. Since collagens are key components of the bladder wall11,12, first we sought to determine the most abundant collagen(s) in the mouse bladder using RNA profiling data sets generated by the Mouse ENCODE project13. Our analysis showed that Collagen 1A1, collagen 3A1, collagen 1A2 and collagen 6A1 are most abundant collagen types within mouse bladder (Figure 2A). We also used the Tabula Muris (a compendium of single cell transcriptome data from mouse (Mus musculus))8 to determine the mRNA expression level of collagens 1, 3, 6 and hyaluronan. The data allow for direct and controlled comparison of gene expression in cell types shared between tissues. This analysis revealed that the expression of these extracellular matrix components is more prevalent in the mesenchymal cell types rather than urothelium (Figure 2B).
Effect of dissociation on viability of isolated cells from bladder 
Flow cytometry analysis demonstrated that enzymatic digestion using the 4 different protocols yielded viability of 83%, 86%, 93% or 90%, respectively. Thus, protocol section 3 was deemed most valuable for preservation of cell viability. We also observed that approximately 4% of the cells were necrotic (PI+/Annexin V-) (Figure 4A). These observations emphasize the efficiency of the digestion protocol and the ensuing benefit on cell viability.
Effect of spinal cord injury on different populations of cells in the bladder 
We observed a significant increase in the total cell number in bladders of SCI mice compared to controls. The pattern of the dot plots obtained from SCI bladders was also slightly different consistent with ongoing organ remodeling due to spinal cord injury (Figure 4B: first column). Compared to controls, the bladders from SCI animals displayed a significant increase in CD45-positive cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61455/61455fig01.jpg" /> 
Figure 1: Representative perfusion completion with lightened color of the liver.&#160;(A) Demonstrates the liver color at the beginning of the perfusion. (B) Shows the lightened liver color at the end of perfusion. The mouse in (A) had spinal cord transection two weeks prior to perfusion resulting in bladder hypertrophy and its protrusion out of the pelvis unlike the mouse in (B) that did not have spinal cord injury; in this case the bladder is small and hidden in the pelvis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61455/61455fig01large.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/61455/61455fig02.jpg" /> 
Figure 2: Transcriptomic expression of extracellular matrix (ECM) components in the mouse bladder.&#160;(A) Bar chart of 43 different collagen types. The expression is stated by Reads Per Kilobase of transcript, per million mapped reads (RPKM) (data is collected from BioProject: PRJNA66167)14. (B) Violin plots of gene expression in cell types obtained from microfluidic droplet-based 3&#8217;-end counting in a pool of male and female dissociated urinary bladder samples (male and females). Counts were log-normalized for each cell using the natural logarithm of 1+ counts per million ln(CPM+1)8. A pseudocount of 1 CPM was added before taking logarithms. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61455/61455fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61455/61455fig03.jpg" /> 
Figure 3: Gating strategy and FMO controls to determine fluorescence spread.&#160;(A) Selection of cell population. (B) Gating strategy for singlets. (C) Gating for necrotic, and early and late apoptotic cells using PI and Annexin V antibody. (D&#8210;F) A schematic dot plot of multicolor flow cytometry (e.g., antibodies conjugated with A, B, C, D fluorochromes + (Annexin V and PI). This shows the fluorescence spread into the antibody with fluorochrome A channel shown by the FMO control compared to an unstained control. Orange dotted line represents FMO gating boundary compared to unstained boundary in red. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61455/61455fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61455/61455fig04.jpg" /> 
Figure 4: Flow cytometry of different cell types in bladder.&#160;(A) Annexin V/PI double staining flow charts. The different combinations of enzymes and chemical used for each protocol are represented in front of the corresponding viability plot. These data demonstrate highest viability was obtained with protocol section 3. (B) Representative histograms illustrating the intensity of Ly-6A/E (Sca-1) and CD326 (Ep-CAM) detected in single channels. (C) Effect of SCI on cell population of the mouse bladder. Upper panel shows results of staining on three dissociated bladders obtained from control non-surgical mice and lower panel shows the results of staining on three animals with SCI. The first column is the total cell population. The second column shows the singlet gating selection. The third column shows the subpopulation of live cells that are negative for B-cells, T-cells and NK cells. The fourth column shows the staining for live cells positive for CD45. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61455/61455fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Amount (for 500 mL)</td>
			<td>Molarity</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>4.091 g</td>
			<td>140 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>0.186 g</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>0.0476 g</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>0.9 g</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1.19 g</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>
Table 1: Components for preparation of Tyrode&#8217;s solution. The indicated components are for preparation of 500 mL Tyrode&#8217;s solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Amount</td>
			<td>Protocol section 1</td>
			<td>Protocol section 2</td>
			<td>Protocol section 3</td>
			<td>Protocol section 4</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>5 mg</td>
			<td>yes</td>
			<td>yes</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>0.03 mM</td>
			<td>yes</td>
			<td>yes</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>132.5 units</td>
			<td>yes</td>
			<td>yes</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Collagenase Type III</td>
			<td>96.4 units</td>
			<td>yes</td>
			<td>yes</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Collagenase Type VI</td>
			<td>50 units</td>
			<td>-</td>
			<td>-</td>
			<td>yes</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>10 units</td>
			<td>yes</td>
			<td>yes</td>
			<td>yes</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>115 units</td>
			<td>-</td>
			<td>-</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Pan Collagenase</td>
			<td>50 units</td>
			<td>-</td>
			<td>-</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>10.5 units</td>
			<td>-</td>
			<td>-</td>
			<td>yes</td>
			<td>yes</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>1.25 units</td>
			<td>yes</td>
			<td>yes</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cell dissociation solution</td>
			<td>1 mL</td>
			<td>yes</td>
			<td>-</td>
			<td>yes</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Recombinant enzyme</td>
			<td>1 mL</td>
			<td>-</td>
			<td>yes</td>
			<td>-</td>
			<td>yes</td>
		</tr>
	</tbody>
</table>
Table 2: Components for preparation of digestion buffer. The indicated components are for preparation of 2.5 mL digestion mix (1 U catalyzes the hydrolysis of 1 &#181;mol a substrate per minute at 37 &#176;C. Refer to the product data sheet for definition of unit of each enzyme).

Watch this Scientific Journal Video about A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury at JoVE.com

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry.

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and ...

a-single-cell-dissociation-approach-for-molecular-analysis-urinary

Nanyang Technological University

Research

JoVE Journal

Biology

5.5K Views.  Boston Children's Hospital. Het doel van dit protocol is om een geoptimaliseerd weefseldissociatieprotocol toe te passen op een muismodel van ruggenmergletsel en de aanpak voor eencellige analyse door stroomcytometrie te valideren.

Video: A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

Medicine

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Cancer Research

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

Immunology and Infection

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.

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 ...

Developmental Biology

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Neuroscience

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (&gt;90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in ...

Efficient Extraction of Astrocytes and Microglia from the Adult Mouse Spinal Cord

Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

Author Spotlight: Isolation of Glial Cells from the Adult Mouse Spinal Cord 

This protocol outlines the isolation of purified astrocytes and microglia from the adult mouse spinal cord, facilitating subsequent applications such as RNA analysis and cell culture. It includes detailed cell dissociation methods and procedures designed to enhance both the quality and yield of isolated cells.

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video