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

10.4K 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.