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.

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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. 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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. 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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://www.jove.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://www.jove.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://www.jove.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://www.jove.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 ...

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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

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 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 ...

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 ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Spinal Cord Transection In Xenopus laevis Tadpoles

Spinal cord injury (SCI) is a permanent affliction, which affects the central nervous system (CNS) motor and sensory nerves, resulting in paralysis beneath the injury site. To date, there is no functional recovery therapy for SCI, and there is a lack of clarity regarding the many complexes and dynamic events occurring after SCI. Many non-mammalian organisms can regenerate after severe SCI, such as teleost fishes, urodele amphibians, and larval stages of anuran amphibians, including Xenopus laevis tadpoles. These are bona fide model organisms to study and understand the response to SCI and the mechanisms underlying successful regenerative processes. This type of research can lead to the identification of potential targets for SCI therapeutic intervention. This article describes how to perform Xenopus laevis tadpole spinal cord transection, including husbandry, surgery, postsurgery care, and functional test evaluation. This injury method can be applied for elucidating the different steps of spinal cord regeneration by studying the cellular, molecular, and genetic mechanisms, as well as histological and functional evolution after SCI and during spinal cord regeneration.

Spinal Cord Transection In Xenopus laevis Tadpoles

Xenopus laevis tadpole spinal cord transection is a relevant injury method to study spinal cord injury and regeneration by making a transverse cut that completely severs the spinal cord at the thoracic level.

Spinal cord injury (SCI) is a permanent affliction, which affects the central nervous system (CNS) motor and sensory nerves, resulting in paralysis ...

An Integrated Approach for Microprotein Identification and Sequence Analysis

Next-generation sequencing (NGS) has propelled the field of genomics forward and produced whole genome sequences for numerous animal species and model organisms. However, despite this wealth of sequence information, comprehensive gene annotation efforts have proven challenging, especially for small proteins. Notably, conventional protein annotation methods were designed to intentionally exclude putative proteins encoded by short open reading frames (sORFs) less than 300 nucleotides in length to filter out the exponentially higher number of spurious noncoding sORFs throughout the genome. As a result, hundreds of functional small proteins called microproteins (&#60;100 amino acids in length) have been incorrectly classified as noncoding RNAs or overlooked entirely.
Here we provide a detailed protocol to leverage free, publicly available bioinformatic tools to query genomic regions for microprotein-coding potential based on evolutionary conservation. Specifically, we provide step-by-step instructions on how to examine sequence conservation and coding potential using Phylogenetic Codon Substitution Frequencies (PhyloCSF) on the user-friendly University of California Santa Cruz (UCSC) Genome Browser. Additionally, we detail steps to efficiently generate multiple species alignments of identified microprotein sequences to visualize amino acid sequence conservation and recommend resources to analyze microprotein characteristics, including predicted domain structures. These powerful tools can be used to help identify putative microprotein-coding sequences in noncanonical genomic regions or to rule out the presence of a conserved coding sequence with translational potential in a noncoding transcript of interest.

The protocol described here provides detailed instructions on how to analyze genomic regions of interest for microprotein-coding potential using PhyloCSF on the user-friendly UCSC Genome Browser. Additionally, several tools and resources are recommended to further investigate sequence characteristics of identified microproteins to gain insight into their putative functions.

Next-generation sequencing (NGS) has propelled the field of genomics forward and produced whole genome sequences for numerous animal species and model ...