The authors wish to thank Dr. Leonid Volkov and &#201;ric Bouchard for their technical advice regarding epifluorescence microscopy.
Financial support 
Funded by the Canadian Institutes of Health Research (grant #MOP-93781) to G.B.

Animal care was in accordance with the Universit&#233; de Sherbrooke animal care and use committee.

1. Tube Preparation
<ol>
	<li>The day before cell sorting, add 1-2 ml of heat-inactivated fetal bovine serum (FBS) to 5 ml polypropylene round bottom tubes, and to 15 ml and 50 ml polypropylene conical tubes. 
	Critical step: Ensure that every tube used in the protocol is coated. 
	Note: FBS coating prevents germ cells from sticking to tube walls.</li>
	<li>Slowly mix O/N by inversion at 4&#160;&#176;C to coat the tubes uniformly using a rotator.</li>
	<li>The next day, remove FBS from coated tubes by decanting.
	<ol>
		<li>*Critical step: Ensure that the 5 ml polypropylene round bottom tubes and the 15 and 50 ml tubes used for cell preparation (protocol steps 2.1 to 2.11) are completely emptied of FBS to prevent fanning (i.e., imprecise deviation of the side streams). Use a P200 pipet to remove the FBS. 
		Note: Residual FBS in tubes prior to sorting may lead to a loss of cells or contamination of other tubes used during cell sorting,&#160;</li>
		<li>Leave a small residual volume of FBS (about 100-200 &#181;l) in the 5 ml polypropylene round bottom tubes used for collecting purified cells.</li>
	</ol>
	</li>
</ol>
2. Cell Preparation
<ol>
	<li>Sacrifice one male mouse under anesthesia using O2/isoflurane (induction at 5% then maintained at 2%) followed by CO2 asphyxiation.</li>
	<li>Excise and decapsulate both testes and mince with small scissors in 500 &#181;l of sorting buffer into a 1.5 ml tube.</li>
	<li>Flush germ cells from seminiferous tubules by gentle up and down pipetting in the 1.5 ml tube, first with a truncated 100-1,000 &#181;l tip, and then with an intact 100-1,000 &#181;l tip.</li>
	<li>Remove debris and clumps by one filtration step using a 40 &#181;m cell strainer and collect filtrate into the 50 ml conical tube previously coated with FBS in step 1.</li>
	<li>Wash the filter once with sorting buffer up to a total volume of 3 ml and transfer filtrate into the 15 ml conical tube previously coated in step 1.</li>
	<li>Add 16 &#181;l of 50 mM EDTA pH 8 per milliliter of cell suspension (48 &#181;l for 3 ml) so as to obtain a final concentration of 0.8 mM EDTA.</li>
	<li>To fix germ cells, slowly add 3 volumes of ice-cold 100% ethanol using a 10 ml pipet with gentle agitation (vortex at low speed), which will produce a milky suspension. 
	Critical step: Slowly adding ethanol is crucial and important to preserve the integrity of the cell preparation. We noted that rapid addition of ethanol causes cell lysis resulting in a major decrease of sorting efficiency. For a 3 ml cell suspension, 9 ml of ethanol should be added in approximately 1 min.</li>
	<li>Incubate cells on ice for 15 min with occasional mixing by inversion.</li>
	<li>Centrifuge cell suspension at 800 x g for 5 min and remove the clear supernatant.</li>
	<li>Gently resuspend the fixed germ cells in 2 ml of sorting buffer.</li>
	<li>Add 4 &#181;l of SYTO16 DNA dye (1 mM solution in DMSO) and incubate for at least 30 min (protected from light). 
	Note: Best sorting results are obtained using SYTO16 since it is a permeable DNA dye that is easily incorporated into the highly compacted nuclei of spermatids.</li>
</ol>
3. Cell Sorter Set Up
Note: Here, a 4-Laser (405 nm - violet, 488 nm - blue, 561 nm - yellow-green, 633 nm - red) 20-parameter BDFACSAria III cell sorter is used. The BD FACSDiva 6.1.3 software is used to visualize and analyze the data. The settings may vary depending on the type of sorter used.
<ol>
	<li>Sterilize the FACS sorter by running 70% ethanol as sheath fluid during the fluidics shutdown procedure (&#34;Cytometer&#34; menu in the software) prior to sorting. Select &#34;Cytometer&#34; in the toolbar of the software and choose &#34;Fluidic shutdown&#34;. Follow the steps mentioned by the processes.</li>
	<li>Prepare and filter 1x PBS with a 0.22 &#181;m filter so as to eliminate any crystals and contaminants. Fill the sheath tank to the upper weld line on the inside of the tank.</li>
	<li>Start the software and log in. Start the FACS sorter to warm up the lasers for at least 30 min prior to sorting. Run two fluidic startup processes to flush out the ethanol from the fluidics. Select &#34;Cytometer&#34; in the toolbar of the software and choose &#34;Fluidic startup&#34;. Follow the steps mentioned by the processes. 
	Note: This restores fluidics with normal sheath fluid (1x PBS).</li>
	<li>Clean the sorting block and the deflection plates with distilled water and dry them thoroughly. Sonicate a 100 &#181;m nozzle placed in a tube of distilled water for 2 min in a sonication bath. Wipe the nozzle thoroughly.</li>
	<li>Insert the 100 &#181;m nozzle into the flow cell and turn the nozzle-locking lever clockwise to the 12 hr position. Set the sheath pressure to 20 psi.</li>
	<li>Turn on the stream and adjust the amplitude to obtain target values for frequency (around 30), Drop 1 (around 150) and Gap (around 12) and to establish a stable droplet breakoff pattern (few satellite drops). Turn off attenuation.</li>
	<li>Make sure that the stream is straight and hits the center of the waste aspirator by changing the angle of the sort block. Note: A 130 &#181;m nozzle at 10 psi was also tested and no obvious difference was found.</li>
	<li>Set laser delay to 0 for the blue, -77.39 for the red, 37.33 for the violet and -39.85 for the yellow-green lasers.</li>
	<li>Set areas scaling to 1.14 for the blue, 1.0 for the red, 0.75 for the violet and 0.96 for the yellow-green lasers.</li>
	<li>Using a Test Sort (in the &#34;Side Stream&#34; window of the software), make sure that side streams are not fanning. In the &#34;Side Stream&#34; window, adjust the voltage sliders of each side stream so that they hit the middle of test collection tube. If the center stream is not tight, adjust the 2nd, 3rd, and 4th drop settings to constrain the center stream.</li>
	<li>Start the Cytometer Setup and Tracking (CS&#38;T) application in the software (&#34;Cytometer&#34; menu).</li>
	<li>Use the cytometer setup and tracking beads at 1 drop per 350 &#181;l to automate the characterization and tracking of the cytometer performance using manufacturer&#39;s instructions.</li>
	<li>Quit the CS&#38;T application. Apply CS&#38;T setting, make sure the stream parameters still allow a stable droplet breakoff pattern, and turn on the Sweet Spot button as described in manufacturer&#39;s instructions (in the &#34;Breakoff window&#34;).</li>
	<li>Optimize the Drop Delay value using fluorescent beads (see Table of Materials).
	<ol>
		<li>Install the collection tubes in the holder. Open the sort block door. In the &#34;Side Stream&#34; window, turn on voltage then select &#34;Test sort&#34; and click the Aspirator Drawer button to open the drawer and have access to the collection tubes.</li>
		<li>Optimize the side streams so they go into the center of the tube. Adjust values and sliders as needed.</li>
		<li>Close the sort block door and make sure to adjust the micrometer dial to obtain the brightest bead spot at the center. Unselect waste drawer, test sort and voltage. Remove the collection tubes.</li>
		<li>Optimize the drop delay according to manufacturers&#39; instructions.
		<ol>
			<li>Briefly, shake bead vial vigorously and dilute 1 drop of beads in 0.5 ml of PBS.</li>
			<li>Using manufacturers&#39; instructions, set the drop delay using the Drop Delay experiment template available in the software. Alternatively, use the Auto Drop Delay feature to set the drop delay.</li>
			<li>Load the tube of fluorescent beads and adjust the flow rate until the threshold rate is ~3,000 to 4,000 events/second. Set the sort precision to &#34;Initial&#34; and finally to &#34;Fine tune&#34; in the &#34;Sort layout&#34; window. Select the optical filter and sort beads. Adjust the Drop Delay until ~100% are sorted to the left. 
			Note: This step is critical to make sure that cells of interest sort into a side stream. The beads and the optical filter are used to achieve close to a 100% droplet deviation.&#160;</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Place a FSC 1.5 ND (neutral density) filter at the left end of the FSC detector block. Set the window extension to 2.00 &#181;s and the FSC Area scaling to 1.00 (in the &#34;Laser&#34; tab in the &#34;Cytometer&#34; window).</li>
	<li>Before loading sample tube, place a sample filter line of 50 &#181;m at the end of the sample line to avoid clumping. To do so, select &#34;Change Sample Filter&#34; in the &#34;Cytometer&#34; menu.</li>
	<li>Test the composition of the sample fluid to achieve good separation without fanning between side stream and to prevent the cells from sticking to the polypropylene tubes.
	<ol>
		<li>Load the sample tube, turn on the deflection plates and select &#34;Test sort&#34; with the drawer closed. Look at side streams if separation occurs without too much fanning. 
		Note: Fanning is likely due to a high protein concentration in the sample buffer.</li>
	</ol>
	</li>
	<li>Load the sample tube into the FACS instrument. Select a sample agitation of 300 rpm and a sample temperature of 4 &#176;C. Select &#34;Acquire Data&#34; in the &#34;Acquisition Dashboard&#34;.</li>
	<li>If necessary, dilute the sample to achieve an appropriate event rate to a maximum of 5,000 events/sec (at a maximum flow rate of 3.0). Strictly respect these limits so as to maintain the purity of the sorted cells.</li>
	<li>To analyze and sort the cells, optimize the FSC threshold value to eliminate debris without interfering with the population of interest. In logarithmic scale, adjust the voltage of the photo-multiplier tube (PMT) detector with the 530/30 filter in order to make sure that the fluorescence signal of the total population fits within the scale, and gate on the positive population (Figure 1, Gate 1).</li>
	<li>Produce a Forward Scatter-area (FSC-A)/Side Scatter-area (SSC-A) plot of the SYTO16 positive population and adjust the FSC-A to around 110 V in linear scale, and the SSC-A to around 150 V in logarithmic scale to visualize all the events while excluding very large cells and cell aggregates.</li>
	<li>In the &#34;Sort Layout&#34; window, set the &#34;Sort Precision Mode&#34; to &#34;4-way purity&#34; (Yield mask: 0, Purity Mask: 32, Phase Mask: 0). Select &#34;Continuous&#34; from the &#34;Target Events&#34; menu for continuous sorting.</li>
	<li>Add populations to sort in the field corresponding to the tubes (in the &#34;Sort Layout&#34; window). Place populations with higher frequencies in the middle and those with lower frequencies at the ends. Set the voltage plates to 2,500 V. During sorting, keep the sample collection tubes on ice.&#160;&#160;&#160;&#160;</li>
</ol>
4. Cell Sorting
<ol>
	<li>Immediately before sorting, filter cells using a 50 &#181;m filter in a previously coated 5 ml polypropylene round bottom tube.</li>
	<li>Wash the filter extensively with 1-2 ml of sorting buffer.</li>
	<li>Sort fixed germ cells using a 488 nm laser-equipped cell sorter following the gating scheme detailed in Figure 1. Collect purified fractions in 5 ml polypropylene round bottom tubes previously coated in section 1 on ice.
	<ol>
		<li>Make sure that 100-200 &#181;l of FBS is kept in the collection tubes to avoid sticking of the sorted cells to the tube wall.</li>
		<li>Make sure that the concentration of SYTO16 is saturating to avoid fluorescence fluctuations during the sorting period. SYTO16 is saturating when there is no further variation of cell staining intensity at the Alexa Fluor 488 parameter (DNA staining) in the sorting graphics. If necessary, add 1 &#181;l of SYTO16 to the sorting tube to reach saturation.</li>
		<li>Display the total events on a graphic displaying the following parameters: number of events vs Alexa Fluor 488-A.</li>
		<li>Select the positive DNA staining cells with Gate 1, as shown in Figure 1.
		<ol>
			<li>Display cells from gate 1 on a graphic using SSC-A vs FSC-A as parameters
			<ol>
				<li>Select the cells with Gate 2, as showed in Figure 1.</li>
				<li>Display cells from Gate 2 on a graphic using FSC-A vs Alexa Fluor 488-A as parameters.</li>
				<li>Select cells with Gate 5 and Gate 6 on Gate 2 graphic, as showed in Figure 1.</li>
				<li>Display separately Gate 5 and Gate 6 on graphics using Alexa Fluor 488-W vs Alexa Fluor 488-A as parameters.</li>
				<li>Select spermiogenesis steps 1-9 spermatids on the Gate 5 graphic and spermiogenesis steps 10-12 spermatids on the Gate 6 graphics, as showed in Figure 1.</li>
			</ol>
			</li>
			<li>Display cells from gate 1 on a graphic using FSC-A vs Alexa Fluor 488-A as parameters
			<ol>
				<li>Select cells with Gate 3 and Gate 4, as showed in Figure 1.</li>
				<li>Display separately Gate 3 and Gate 4 on graphics using Alexa Fluor 488-W vs Alexa Fluor 488-A as parameters.</li>
				<li>Select spermiogenesis steps 13-14 spermatids on the Gate 3 graphic and spermiogenesis steps 15-16 spermatids on the Gate 4 graphic, as showed in Figure 1.&#160;&#160;&#160;&#160;</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+actin+and+myosin+during+spermatogenesis.">The role of actin and myosin during spermatogenesis.</a> Mol. Biol. Rep. 38 (6), 3993-4001 (2011).</li><li>Cheng, C. Y., Mruk, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+junction+dynamics+in+the+testis:+Sertoli-germ+cell+interactions+and+male+contraceptive+development.">Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development.</a> Physiol. 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Reprod. 14 (2), 388-394 (1999).</li></ol>

The authors declare no competing financial interests.

Spermatogenic cells have always been challenging to study given the complexity of the seminiferous epithelium, as well as the limited success of in vitro culture. Over the years, many approaches to purify germ cells from various species were developed. Sedimentation techniques using gravity purification with Percoll or bovine serum albumin gradients usually provide a good yield of intact germinal cells, but lack proper definition between some cell types such as meiotic tetraploid cells and spermatids10</sup...

Haploid round spermatids differentiate into spermatozoa by a process called spermiogenesis. This involves many different steps including the acquisition of a flagellum, chromatin and cytoskeleton remodeling, condensation of the nucleus as well as the loss of most of the cytoplasm. These unique cellular events must be finely regulated in order to produce a mature functional gamete with an intact genome suitable for fertilization. Spermiogenesis can hardly be studied in vitro since no reliable cell culture system has so far been able to support progression through the different steps of the process. Moreover, actual in vitro techniques lead to a poor yield1,2. In vivo, proper transitions through the different steps of spermiogenesis are crucial for the natural functional integrity of the male gamete. Successful purification of spermatids according to their differentiation steps has never been accomplished with a level of enrichment sufficient to allow molecular characterization of spermiogenesis. For instance, purification of key steps of the spermatidal differentiation would be especially useful to study the developing acrosome, formation of the midpiece3, cell junction dynamics4, RNA dynamics5, chromatin remodeling process6,7 or genomic stability8. Purification of spermatids has been hampered by their progressive morphological transformation, the lack of known stage-specific external biomarkers, and their peculiar shape and size. 
Although most male germ cells display a direct relationship between DNA staining and ploidy (DNA content), we noticed that such positive correlation is no longer applicable to spermatids. This stems from our early observation that seminiferous tubule sections show variable intensity of DNA staining throughout the different spermiogenesis steps. Although DNA staining is consistent with their haploid set of chromosomes from spermiogenesis steps 1 to 7 (round spermatids), we observed a sharp increase in fluorescence intensity with DAPI or SYTO 16 around the onset of nuclear reorganization and chromatin remodeling (spermiogenesis step 8) reaching a peak at the onset of nuclear condensation (spermiogenesis steps 11-12). Following condensation of the nucleus, DNA staining intensity decreases until spermiation (spermiogenesis step 16). We surmised that this was likely associated with the formation of their peculiar chromatin structure transition where histones are replaced by protamines. We therefore developed a reliable flow cytometry method that allows the separation of spermatids using the variation of DNA intensity of spermatids as a main selection parameter.
A simple flow cytometry approach is described to separate mouse spermatids with high purity (95-100%) based on their apparent DNA content (SYTO16 staining), size and granulosity. Spermatids are separated into four populations; spermiogenesis steps 1-9, 10-12, 13-14 and 15-16. Purified spermatids are suitable for genetic/genomic analysis, as well as proteomic applications as described in a recent publication from our group9.

<table border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Isoflurane</td>
			<td style="width:119px;">ABBOT</td>
			<td style="width:129px;">05260-05</td>
			<td style="width:184px;">For mouse anesthesia before euthanasia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Fetal bovine serum</td>
			<td style="width:119px;">Wisent</td>
			<td style="width:129px;">90150</td>
			<td style="width:184px;">For tube coating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">1X PBS</td>
			<td style="width:119px;"></td>
			<td style="width:129px;"></td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">EDTA</td>
			<td style="width:119px;">BioShop</td>
			<td style="width:129px;">EDT</td>
			<td style="width:184px;">For sorting buffer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">HEPES</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:129px;">H</td>
			<td style="width:184px;">For sorting buffer preparation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">100 % Ethanol</td>
			<td style="width:119px;">Les alcools de commerce</td>
			<td style="width:129px;">092-09-11N</td>
			<td style="width:184px;">For cell fixation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">SYTO 16</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:129px;">S7578</td>
			<td style="width:184px;">DNA staining</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">5 ml polypropylene round bottom tubes</td>
			<td style="width:119px;">BD Falcon</td>
			<td style="width:129px;">352063</td>
			<td style="width:184px;">Sorted cells collection</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">15 ml polypropylene conical bottom tubes</td>
			<td style="width:119px;">PROgene</td>
			<td style="width:129px;">1500</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:135px;">50 ml polypropylene conical bottom tubes</td>
			<td style="width:119px;">PROgene</td>
			<td style="width:129px;">5000</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">TEC4 anaesthetic vaporizer</td>
			<td style="width:119px;">Ohmeda</td>
			<td style="width:129px;">1160526</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:135px;">CO2 gas tank</td>
			<td style="width:119px;">Praxair</td>
			<td style="width:129px;">C799117902</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:135px;">O2 gas tank</td>
			<td style="width:119px;">Praxair</td>
			<td style="width:129px;">O254130501</td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">Homemade mouse gas chamber</td>
			<td style="width:119px;"></td>
			<td style="width:129px;"></td>
			<td style="width:184px;">For mouse euthanasia</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">40 &#181;m Falcon cell strainer</td>
			<td style="width:119px;">Corning Incorporated</td>
			<td style="width:129px;">352340</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">50-micron sample line filters</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">649049</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">Vortex mixer</td>
			<td style="width:119px;">Labnet international, inc.</td>
			<td style="width:129px;">S0200</td>
			<td style="width:184px;">For cell fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Dynac centrifuge</td>
			<td style="width:119px;">Clay Adams</td>
			<td style="width:129px;">101</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:135px;">Celltrics 50 &#181;m filters</td>
			<td style="width:119px;">Partec</td>
			<td style="width:129px;">04-004-2327</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:135px;">488 nm laser-euipped cell sorter</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">FACSAria III</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:135px;">Accudop Fluorescent Beads</td>
			<td style="width:119px;">BD Biosciences</td>
			<td style="width:129px;">345249</td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:135px;">Sorting Buffer: 1X PBS, 1mM EDTA pH 8.0, 25mM HEPES pH 7.0, 1%FBS</td>
			<td></td>
			<td></td>
			<td style="width:184px;">FBS is heat-inactivated. Make fresh solution, 0.22 &#956;m filtered and keep at 4&#176;C.</td>
		</tr>
	</tbody>
</table>

step-specific sorting of mouse spermatids by flow cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

Gating strategy used with flow cytometry
Figure 1 represents the gating strategy used in flow cytometry to sort four highly pure spermatid populations. Briefly, cells with positive DNA staining (Alexa Fluor 488-A) are first selected with Gate 1. Spermatids from spermiogenesis steps 1-12 are selected (Gate 2) on a dot plot showing the granulosity (SSC-A) vs size (FSC-A) from Gate 1. Then, spermatids from spermiog...

Watch this Scientific Journal Video about Step-specific Sorting of Mouse Spermatids by Flow Cytometry at JoVE.com

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

step-specific-sorting-of-mouse-spermatids-by-flow-cytometry

Research

JoVE Journal

Developmental Biology

10.5K Views.  Université de Sherbrooke. We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

Video: Step-specific Sorting of Mouse Spermatids by Flow Cytometry

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis (which is the branching off of major vessels) and vasculogenesis (de novo vessel formation), perfusion mapping techniques such as resin casts, in vivo ultrasound imaging, and micro-dissection have been limited in demonstrating the intimate relationships between these two processes and developing renal structures within the embryo. Here, we describe the procedure of in utero intra-cardiac ultrasound-guided FITC-labeled tomato lectin microinjections on mouse embryos to gauge the ontogeny of renal perfusion. Tomato lectin (TL) was perfused throughout the embryo and kidneys harvested. Tissues were co-stained for various kidney structures including: nephron progenitors, nephron structures, ureteric epithelium, and vasculature. Starting at E13.5 large caliber vessels were perfused, however peripheral vessels remained unperfused. By E15.5 and E17.5, small peripheral vessels as well as glomeruli started to become perfused. This experimental technique is critical for studying the role of vasculature and blood flow during embryonic development.

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the development of hypertension during obesity. The extent of hypertrophy and hyperplasia depends on depot location, sex, and the type of Adipocyte Progenitor Cell (APC) phenotypes present. Techniques used for APC and preadipocytes isolation in the last 10 years have drastically improved the accuracy at which individual cells can be identified based on specific cell surface markers. However, isolation of APC and adipocytes can be a challenge due to the fragility of the cell, especially if the intact cell must be retained for cell culture applications.
Magnetic-activated Cell Sorting (MCS) provides a method of isolating greater number of viable APC per weight unit of adipose tissue. APC harvested by MCS can be used for in vitro protocols to expand preadipocytes and differentiate them into adipocytes through use of growth factor cocktails allowing for analysis of the prolific and adipogenic potential retained by the cells. This experiment focused on the aortic and mesenteric PVAT depots, which play key roles in the development of cardiovascular disease during expansion. These protocols describe methods to isolate, expand, and differentiate a defined population of APC. This MCS protocol allows isolation to be used in any experiment where cell sorting is needed with minimal equipment or training. These techniques can aid further experiments to determine the functionality of specific cell populations based on the presence of cell surface markers.

Here we report a method for isolation of Adipocyte Progenitor Cell (APC) populations from Perivascular Adipose Tissue (PVAT) using Magnetic-activated Cell Sorting (MCS). This method allows for an increased isolation of APC per gram of adipose tissue when compared to Fluorescence-Activated Cell Sorting (FACS).

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

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

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

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

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

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.

We describe the use of hematopoietic stem cell transplantation (HSCT) to assess the malignant potential of genetically engineered hematopoietic cells. HSCT is useful for evaluating various malignant hematopoietic cells in vivo as well as generating a large cohort of mice with myelodysplastic syndromes (MDS) or leukemia to evaluate novel therapies.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...