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 &#956;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 &#181;L)</td>
			<td>VWR</td>
			<td>89049-168</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerosol Filter Wide Orifice Pipettor Tips (1000 &#181;L)</td>
			<td>VWR</td>
			<td>89049-168</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD326 (Ep-CAM), rat monoclonal, IgG2a, &#954;, affinity purified</td>
			<td>BioLegend</td>
			<td>118213</td>
			<td></td>
		</tr>
		<tr>
			<td>BB515 Rat Anti-Mouse CD45, rat monoclonal, IgG2b, &#954;, 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&#34; 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&#34; 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 &#38; 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, &#954;, 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 &#181;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&#34; 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, &#954;, affinity purified</td>
			<td>BioLegend</td>
			<td>115509</td>
			<td>Dump Channel</td>
		</tr>
		<tr>
			<td>PE/Cy5 anti-mouse CD3&#949; 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, &#954;, 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, &#954;, 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, &#954;, 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, &#954;, 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, &#954;, 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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

5.4K Views.  Boston Children's Hospital. 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.

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

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

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

Summary

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Chapters in this video

Molecular Evolution of the Tre Recombinase

Spinal Cord Electrophysiology

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Single-cell Microinjection for Cell Communication Analysis

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Single-cell Microfluidic Analysis of Bacillus subtilis

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation