Name: Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation (Video) | JoVE Journal
Uploaded: 2013-10-09T21:20:00.000+00:00
Duration: 9 min 48 s
Description: Learn STA-PUT velocity sedimentation to separate spermatogenic cell types from mouse testes based on size and density. This method enables isolation of large quantities of pure cell populations using simple glassware and gravity for reproductive biology research.

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5 minutes

This research was supported by NIH grants GM055360 to SLB and U54HD068157 to RGM. JMB was supported by the T32 Genetics Training Grant at the University of Pennsylvania (GM008216).

The STA-PUT protocol involves three stages: 1) Set up of the apparatus and reagents, 2) Preparation of cell suspension from whole testes, and 3) Cell loading, sedimentation, and fraction collection. When performed by a team of two researchers, the protocol takes eight hours on average.
1. Setting up the STA-PUT Apparatus (Figure 1)
***STA-PUT apparatus should be placed in a 4&#176;C large refrigerator or a cold room that can also accommodate a fraction collector, if that method of collection is preferred.
<ol>
	<li>The night before (or at least a few hours before) you perform the method, wash all equipment (especially the glassware and tubing) and sterilize with 70% ethanol. Let equipment dry completely before assembling the apparatus as illustrated in Figure 1.</li>
	<li>Secure the two 2 L cylinders (Figures 1B and C) and the cell loading chamber (Figure 1A) to the top platform and connect all with two small pieces of tubing with tube clamps. Clamp all tubes closed. Seal the spout on the right-most 2 L cylinder.</li>
	<li>Place a small stir bar in the cell loading chamber (Figure 1A) and a larger stir bar in the left-most 2 L cylinder (Figure 1B) that will contain the 2% BSA.</li>
	<li>Place the 2 L sedimentation chamber on the platform (Figure 1D). Place the metal baffle (Figure 1F) directly on top of the opening in the bottom of the sedimentation chamber (Figure 1D). This is critical, as the baffle prevents vortexing of the liquid and disruption of the cell gradient during fraction collection. Place the lid on top of the sedimentation chamber.</li>
	<li>After applying a very small amount of vacuum grease to the ground glass joint of the three-way stopcock (Figure 1G), clamp the stopcock to the bottom of the sedimentation chamber, connecting the ground glass joints of the stopcock and the sedimentation chamber.</li>
	<li>Connect the cell-loading chamber (Figure 1A) to the right outlet of the stopcock with tubing. Close the stopcock.</li>
	<li>Attach the cell fractionation tubing to the left outlet of the stopcock. The fractionation tubing comprises a piece of tubing with a glass Pasteur pipette connected to the open end. A piece of smaller bore tubing is attached to the narrow end of the glass pipette. The narrow pipette restricts the flow of the cell suspension during fraction collecting. Clamp this small tube at the very bottom.</li>
	<li>Prepare 2 L Krebs (1x) buffer the day of the experiment (Table 1). Then, prepare 550 ml 2% BSA in 1x Krebs, 550 ml 4% BSA in 1x Krebs, and 50 ml 0.5% BSA in 1x Krebs. Filter and cool these solutions to 4 &#176;C.</li>
	<li>Pour the 4% BSA solution in the right 2 L cylinder (Figure 1C) and the 2% BSA solution in the left 2 L cylinder (Figure 1B). Make sure there are no large bubbles in the tubing that connects these cylinders.</li>
	<li>Pour Krebs buffer into the cell loading chamber and fill the tubing connecting this chamber with the sedimentation chamber, making sure there are no large bubbles, as these could disturb the gradient. Squeezing or flicking the tubing gently helps to remove the bubbles. Make sure all of the Krebs buffer is in the tube and not in the cell chamber.</li>
	<li>Allow a small amount of Krebs buffer to flow into the sedimentation chamber and then drain almost all the buffer into the cell fractionation tubing in order to fill the tube and remove any large bubbles.</li>
	<li>Place the fraction collector directly under the sedimentation chamber. Make sure that all the fraction tubes are in place and covered with plastic wrap to prevent contamination.</li>
</ol>
2. Isolating Spermatogenic Cells from Whole Testes
<ol>
	<li>Dissect testes from ca12 adult mice (preferably at least eight weeks old) and place in Krebs buffer at room temperature to wash off any contaminating material. This protocol being demonstrated involves the use of laboratory mice and was executed in compliance with the guidelines of the University of Pennsylvania Institutional Animal Care and Use Committee.</li>
	<li>Add 45 mg collagenase to 50 ml Krebs buffer in a conical tube right before you intend to add the seminiferous tubules. Allow this solution to heat up to 33 &#176;C for a few minutes. Split evenly into two 50 ml conical tubes.</li>
	<li>Decapsulate the testes in a separate plate containing 8 ml Krebs buffer, discarding the tunica albuginea and releasing the seminiferous tubules. The tunica albuginea is the thin membrane that surrounds the seminiferous tubules. The tubules should readily detach from the tunica albuginea. To do this, make an incision in the tunica albuginea, hold this membrane with a pair of forceps, and push the tubules out of and away from the membrane with another pair of forceps.</li>
	<li>Add 4 ml Krebs buffer containing the tubules to each conical tube containing 25 ml of collagenase solution and incubate, shaking, at 33 &#176;C for 10 min. At the end, the tubules should have a "spaghetti-like" appearance.</li>
	<li>Allow the tubules to settle for ~5 min to the bottom of the tube. Pour out the supernatant and wash 2x in 25 ml Krebs buffer (at room temperature), allowing the tubules to settle to the bottom of the tube each time. Leave ~5 ml Krebs buffer in each tube.</li>
	<li>While washing, add 30 mg trypsin to 50 ml Krebs buffer in a falcon tube right before you intend to add the seminiferous tubules. Allow this solution to heat up to 33 &#176;C for a few minutes. Split evenly into two 50 ml conical tubes.</li>
	<li>Add 25 ml trypsin solution to each of the two tubes containing the tubules. Add 3 &#956;g DNAse (1 &#956;g/10ml) to each tube to prevent cells from clumping. Incubate, shaking, at 33 &#176;C for 10 min.</li>
	<li>Use a wide bore pipette to agitate the solution containing the tubules, pipetting them in and out approximately 10x. The solution should begin to look more like a single cell suspension.</li>
	<li>Incubate, shaking, at 33 &#176;C for an additional 10 min. Use a wide bore pipette to disperse the tubules into a single cell suspension, pipetting them in and out approximately 25x. If you still see a lot of tubules or cell clumps, disperse more with the pipette and/or add another 3 &#956;g DNAse to each tube (double initial concentration).</li>
	<li>Filter the single cell suspension through a 100 &#956;m mesh cell strainer (one for each tube of 30 ml cell suspension). Combine the cell suspensions and count the total number of cells (should be between 7-8 x 108 cells). ***DO NOT LOAD MORE THAN 800,000,000 cells onto the gradient.</li>
	<li>Based on the cell count obtained in step 2.10, pellet the appropriate volume of filtered cells at 450 rcf for 5 min and wash with 30 ml Krebs buffer. Repeat. ***Usually 22 testes will yield more than 800,000,000 cells, but do not load more than this number of cells.</li>
	<li>Carefully resuspend cells in 25 ml 0.5% BSA containing between 2.5 and 5 &#956;g DNAse (depending on the degree of cell clumping) without creating bubbles. Make sure the cells do not clump. Filter the single cell suspension through a 100 &#956;m mesh cell strainer. The cells are now ready to load. Mix well before loading by inverting the tube a few times.</li>
</ol>
3. Cell Loading and Sedimentation
<ol>
	<li>Make sure that the stopcock is closed, but in the position that will allow liquid to flow from the cell chamber (Figure 1A) into the sedimentation chamber (Figure 1D).</li>
	<li>Turn both stir bar plates on to a low setting (approximately 70 rpm) to allow the stir bars to move continuously, but slowly.</li>
	<li>Pour the cell suspension into the cell chamber (Figure 1A) and open the stopcock so that the cells flow slowly into the sedimentation chamber at a rate of 10 ml/min (Figure 1D). Close the stopcock. Flow rate can be determined by marking volume intervals on the sedimentation chamber.</li>
	<li>Pour 5 ml of 0.5% BSA solution into the cell chamber and drain into the sedimentation chamber at a rate of 10 ml/min. Close the stopcock. This step will wash the cells out of the cell chamber. Repeat 4x.</li>
	<li>Prepare the gradient: Open the clamps between the cell chamber (Figure 1A) and the two 2 L cylinders (Figure 1B and C), and begin to drain the liquid into the sedimentation chamber at a rate of 40 ml/min (Figure 1D) immediately. Adjust the flow rate so it takes approximately 20-30 min to load the BSA gradient into the sedimentation chamber. A thin, undisturbed layer of cells lying on top of the BSA gradient should be visible. Once most of the BSA is loaded, close the stopcock and turn it to the position that will allow liquid to drain from the sedimentation chamber into the fractionation tube.</li>
	<li>Turn off the stir plates.</li>
	<li>Allow the cells to sediment for one hour and 45 minutes. DO NOT DISTURB THE APPARATUS DURING THIS TIME. Any mechanical agitation could disturb the gradient.</li>
</ol>
4. Fractionation and Analysis of Fractions
***As you perform the following steps, be careful not to disturb the gradient. If the BSA gradient is disturbed, the procedure will not work!
<ol>
	<li>Once the cells have sedimented, slowly drain 50 ml from the sedimentation chamber into a 50 ml conical tube and set aside. This fraction usually contains unwanted clumps of cells and debris.</li>
	<li>Attach the fraction tube to the fraction collector. ***It is also possible to collect the fractions by hand if a fraction collector is not available.</li>
	<li>Collect fractions: Adjust the flow rate with the stopcock so that 10 ml (filling one collection tube) is collected every 45 sec.</li>
	<li>Cap the fraction collection tubes and spin for 5 min at 450 rcf at 4 &#176;C. Pour off supernatant gently, resuspend cells in remaining liquid, and keep cells on ice.</li>
	<li>Once all fractions are collected, microscopically analyze the fractions for different cell populations. Different cell types can be distinguished based on cell size and nuclear morphology8,15 . Figure 2 illustrates "representative results" for the three pooled fractions that are enriched for (a) somatic and meiotic cells and spermatogonia, (b) round spermatids, and (c) elongating and condensing spermatids.
	<ul>
		<li>Type A spermatogonia are 12-14&#956;m ca. in diameter and have round nuclei that are homogenous in chromatin composition.</li>
		<li>Type B spermatogonia are 8-9&#956;m ca. in diameter and have round nuclei that show more dense heterochromatin along the nuclear periphery.</li>
		<li>Pre-leptotene spermatocytes are 7.5-8.2&#956;m ca. in diameter, have less cytoplasm than Type B spermatogonia, and have nuclei that look similar to those in Type B spermatogonia.</li>
		<li>Leptotene primary spermatocytes are 8-10 &#956;m ca. in diameter and have nuclei that look similar to those of pre-leptotene spermatocytes, but seem to gain more dense chromatin at the nuclear membrane.</li>
		<li>Zygotene primary spermatocytes are 10-12 &#956;m ca. in diameter and have nuclei that look similar to those of leptotene primary spermatocytes.</li>
		<li>Pachytene spermatocytes are anywhere from 12-18 &#956;m ca. in diameter and consist of a thin rim of cytoplasm surrounding a large nucleus. More clumps of dense chromatin can be seen in the nucleus.</li>
		<li>Round spermatids are 10 &#956;m ca. in diameter and have a round nucleus with a densely-staining chromocenter in the middle.</li>
		<li>Residual bodies are ~6.5 &#956;m in diameter, are round, and lack a nucleus.</li>
		<li>Elongating and condensing spermatids are similar in size to round spermatids, but have a unique, sickle-shaped nucleus.</li>
	</ul>
	<ol>
		<li>Start with fraction 15 and analyze every five fractions up to 85. Usually, fractions below 15 will be too mixed and clumpy, and fractions above 85 will contain few to no usable cells.</li>
		<li>To analyze a fraction, take 5uL of liquid from the fraction collection tube (once cells have been resuspended after centrifugation) and add to 5uL of 8% formaldehyde in Krebs buffer. Allow the fixed cells to sit at room temperature for five minutes.</li>
		<li>Add 5uL of 0.1% Triton and DAPI (5 &#956;/ml Krebs buffer) to the fixed cells. Allow the cells to sit at room temperature for 5 min.</li>
		<li>Place 10 &#956;l of the resulting solution onto a slide, cover with a cover slip, and analyze with a fluorescence microscope to determine the purity of each fraction.</li>
		<li>Combine fractions that are similar in size and nuclear morphology (see "Representative Results") to create the following populations of cells: meiotic and somatic diploid cells, round spermatids, and condensing/elongating spermatids.</li>
	</ol>
	</li>
</ol>

<ol><li>Wistuba, J., Stukenborg, J., Luetjens, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mammalian+Spermatogenesis%5BTitle%5D)+AND+%22Funct.+Dev.+Embryol%22%5BJournal%5D)">Mammalian Spermatogenesis.</a> Funct. Dev. Embryol. 1, 99-117 (2007).</li>
<li>Hess, R. A., Cooke, P. S., Hofmann, M. C., Murphy, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanistic+insights+into+the+regulation+of+the+spermatogonial+stem+cell+niche%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">Mechanistic insights into the regulation of the spermatogonial stem cell niche.</a> Cell Cycle. 5, 1164-1170 (2006).</li>
<li>Dores, C., Alpaugh, W., Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+in+vitro+culture+to+in+vivo+models+to+study+testis+development+and+spermatogenesis%5BTitle%5D)+AND+%22Cell+Tissue+Res%22%5BJournal%5D)">From in vitro culture to in vivo models to study testis development and spermatogenesis.</a> Cell Tissue Res. 349, 691-702 (2012).</li>
<li>Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S., Saitou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reconstitution+of+the+mouse+germ+cell+specification+pathway+in+culture+by+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.</a> Cell. 146, 519-532 (2011).</li>
<li>Easley, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+differentiation+of+human+pluripotent+stem+cells+into+haploid+spermatogenic+cells%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells.</a> Cell Rep. 2, 440-446 (2012).</li>
<li>Bellve, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification%2C+culture%2C+and+fractionation+of+spermatogenic+cells%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Purification, culture, and fractionation of spermatogenic cells.</a> Methods Enzymol. 225, 84-113 (1993).</li>
<li>Meistrich, M. L., Bruce, W. R., Clermont, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+composition+of+fractions+of+mouse+testis+cells+following+velocity+sedimentation+separation%5BTitle%5D)+AND+%22Exp.+Cell+Res%22%5BJournal%5D)">Cellular composition of fractions of mouse testis cells following velocity sedimentation separation.</a> Exp. Cell Res. 79, 213-227 (1973).</li>
<li>Romrell, L. J., Bellve, A. R., Fawcett, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+mouse+spermatogenic+cells+by+sedimentation+velocity.+A+morphological+characterization%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization.</a> Dev. Biol. 49, 119-131 (1976).</li>
<li>Miller, R. G., Phillips, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+cells+by+velocity+sedimentation%5BTitle%5D)+AND+%22J.+Cell+Physiol%22%5BJournal%5D)">Separation of cells by velocity sedimentation.</a> J. Cell Physiol. 73, 191-201 (1969).</li>
<li>Bastos, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometric+characterization+of+viable+meiotic+and+postmeiotic+cells+by+Hoechst+33342+in+mouse+spermatogenesis%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.</a> Cytometry A. 65, 40-49 (2005).</li>
<li>Getun, I. V., Torres, B., Bois, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry+purification+of+mouse+meiotic+cells%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Flow cytometry purification of mouse meiotic cells.</a> J. Vis. Exp. (50), e2602(2011).</li>
<li>Grabske, R. J., Lake, S., Gledhill, B. L., Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Centrifugal+elutriation%3A+separation+of+spermatogenic+cells+on+the+basis+of+sedimentation+velocity%5BTitle%5D)+AND+%22J.+Cell+Physiol%22%5BJournal%5D)">Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.</a> J. Cell Physiol. 86, 177-189 (1975).</li>
<li>Meistrich, M. L., Trostle, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+mouse+testis+cells+by+equilibrium+density+centrifugation+in+renografin+gradients%5BTitle%5D)+AND+%22Exp.+Cell+Res%22%5BJournal%5D)">Separation of mouse testis cells by equilibrium density centrifugation in renografin gradients.</a> Exp. Cell Res. 92, 231-244 (1975).</li>
<li>Gassei, K., Ehmcke, J., Schlatt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+enrichment+of+undifferentiated+GFR+alpha+1%2B+spermatogonia+from+immature+rat+testis+by+magnetic+activated+cell+sorting%5BTitle%5D)+AND+%22Cell+Tissue+Res%22%5BJournal%5D)">Efficient enrichment of undifferentiated GFR alpha 1+ spermatogonia from immature rat testis by magnetic activated cell sorting.</a> Cell Tissue Res. 337, 177-183 (2009).</li>
<li>Bellve, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spermatogenic+cells+of+the+prepuberal+mouse.+Isolation+and+morphological+characterization%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization.</a> J. Cell Biol. 74, 68-85 (1977).</li>
<li>Handel, M. A., Schimenti, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetics+of+mammalian+meiosis%3A+regulation%2C+dynamics+and+impact+on+fertility%5BTitle%5D)+AND+%22Nat.+Rev.+Genet%22%5BJournal%5D)">Genetics of mammalian meiosis: regulation, dynamics and impact on fertility.</a> Nat. Rev. Genet. 11, 124-136 (2010).</li>
<li>Zhao, M., Shirley, C. R., Mounsey, S., Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nucleoprotein+transitions+during+spermiogenesis+in+mice+with+transition+nuclear+protein+Tnp1+and+Tnp2+mutations%5BTitle%5D)+AND+%22Biol.+Reprod%22%5BJournal%5D)">Nucleoprotein transitions during spermiogenesis in mice with transition nuclear protein Tnp1 and Tnp2 mutations.</a> Biol. Reprod. 71, 1016-1025 (2004).</li>
<li>Manku, G., Mazer, M., Culty, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neonatal+testicular+gonocytes+isolation+and+processing+for+immunocytochemical+analysis%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Neonatal testicular gonocytes isolation and processing for immunocytochemical analysis.</a> Methods Mol. Biol. 825, 17-29 (2012).</li>
<li>Dehnugara, T., Dhar, S., Rao, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+in+vitro%2C+short-term+culture+method+for+mammalian+haploid+round+spermatids+amenable+for+molecular+manipulation%5BTitle%5D)+AND+%22Mol.+Reprod.+Dev%22%5BJournal%5D)">An in vitro, short-term culture method for mammalian haploid round spermatids amenable for molecular manipulation.</a> Mol. Reprod. Dev. 79, 19-30 (1002).</li>
</ol>

The authors declare that they have no competing financial interests. This protocol being demonstrated involves the use of laboratory mice and was executed in compliance with all relevant guidelines, regulations and regulatory agencies. Animals used in this protocol are maintained under the guidance and approval of the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC protocol #804284).

Those who study spermatogenesis rely on animal models for spermatogenic cell samples, as a reliable cell culture system does not yet exist for generating all spermatogenic cell types3. Although spermatogenic cells are readily collected from whole testes, only a mixed population results. This poses a problem for those who wish to study specific subtypes of these cells, such as meiotic cells, round spermatids, and condensing spermatids. Three different methods are currently used to separate these subtypes of ...

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes1. Briefly, stem-like spermatogonia that reside near the epithelium of the seminiferous tubule divide and differentiate into spermatocytes, which then undergo meiotic divisions. After meiosis is complete, the resulting haploid cells, or round spermatids, undergo spermiogenesis, a differentiation process that involves the shedding of cytoplasm and compaction of the nucleus. Spermatids gradually develop a flagellum and undergo elongation and condensation of the nucleus, producing elongating and then condensing spermatids, respectively. The end products are spermatozoa, which are released into the lumen of the seminiferous tubule and ultimately into the epididymis where they mature further.
Because the process of spermatogenesis relies on special hormonal and molecular conditions in the testes, a reliable in vitro culture system for the entire process of spermatogenesis has not yet been developed2,3. Culture methods have been developed for creating "primordial germ cell-like cells" and haploid, "round spermatid-like cells" from stem cells, but these methods are not yet able to generate large numbers of these cells and fail to produce later spermatogenic cell types4,5. Fortunately, the spermatogenic cell types differ significantly in size, which allows for a single-cell suspension obtained from whole testes to be separated with a liquid gradient. The STA-PUT method, demonstrated here, uses a linear BSA gradient and simple sedimentation to separate spermatogenic cells based on size and mass6-9.
The STA-PUT method has several advantages over the other two most widely used methods to separate spermatogenic cell types: FACS and elutriation10-13. The STA-PUT apparatus requires only several pieces of specialized glassware assembled in a cold room or large refrigerator. Thus, it is less expensive than using a cell sorter or an elutriator. The STA-PUT method yields higher amounts of cells per cell type and testis than can be sorted by FACS in a comparable time frame, although the purity of each cell population is not as high as those obtained with FACS11. Cell sorting utilizing magnetic beads (magnetic activated cell sorting, MACS) has recently been successfully employed for enrichment of spermatogonia from a mixed testicular cell population, but it is currently unsuitable for separating spermatocytes or spermatids due to lack of knowledge of appropriate surface markers14. An additional advantage of the STA-PUT method over FACS or MACS is the ability to isolate viable cells suitable for subsequent culture because, in contrast to most FACS protocols, it does not require any DNA or other types of staining. For studies that require large yields of spermatogenic cells types at ~90% purity, the STA-PUT is an ideal method.

<table><tbody><tr><td>BSA</td><td>Affymetrix/USB</td><td>10857 100MG</td><td>Alternate can be used.</td></tr><tr><td>0.5%, 2%, and 4% BSA solutions</td><td>Dissolve 0.25 g, 11 g, and 22 g (respectively) in total volumes of 50 ml, 550 ml, and 550 ml of 1x KREBS (respectively).</td><td>To be made day of the STA-PUT procedure. Filter sterilize.</td><td></td></tr><tr><td>KREBS (10x)</td><td>3.26 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; + 139.5 g NaCl +5.89 g MgSO&lt;sub&gt;4&lt;/sub&gt;/7H&lt;sub&gt;2&lt;/sub&gt;0 + 40 g Dextrose + 3.78 g CaCl&lt;sub&gt;2&lt;/sub&gt;/2H&lt;sub&gt;2&lt;/sub&gt;0 + 7.12 g KCl, bring to 2 L with ddH&lt;sub&gt;2&lt;/sub&gt;0</td><td>Autoclave/filter and store at 4 &amp;deg;C for several months.</td><td></td></tr><tr><td>KREBS (1x)</td><td>Dissolve 4.24 g NaHCO&lt;sub&gt;3 &lt;/sub&gt;in 100 ml ddH&lt;sub&gt;2&lt;/sub&gt;0. Add 200 ml 10x KREBS. Bring to 2 L with ddH&lt;sub&gt;2&lt;/sub&gt;0.</td><td>To be made day of the STA-PUT procedure. Filter sterilize.</td><td></td></tr><tr><td>Collagenase</td><td>Sigma</td><td>C9891-1G</td><td> </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>DNAse</td><td>Sigma</td><td>DNEP-5MG</td><td> </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T9201-1G</td><td> </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>DAPI</td><td>Preference of researcher</td><td></td><td></td></tr><tr><td>Triton-X100</td><td>Preference of researcher</td><td></td><td></td></tr><tr><td>Formaldehyde (~37%)</td><td>Preference of researcher</td><td></td><td></td></tr><tr><td> </td><td></td><td></td><td></td></tr><tr><td>TABLE 2: EQUIPMENT</td><td></td><td></td><td></td></tr><tr><td> </td><td></td><td></td><td></td></tr><tr><td>Equipment</td><td>Company</td><td>Catalog #</td><td>Comments</td></tr><tr><td>Complete STA-PUT apparatus</td><td>ProScience Glass Shop</td><td>STA-PUT (this procedure uses the standard sedimentation chamber: Cat. No. 56700-500)</td><td>Includes all glassware and equipment for the apparatus. Alternate glassware is available for performing STA-PUT with smaller cell numbers.</td></tr><tr><td>10 &amp;mu;m mesh filter</td><td>Fisherbrand</td><td>22363549</td><td> </td></tr><tr><td>Mouse dissection tools</td><td>Preference of researcher</td><td></td><td></td></tr><tr><td>14 ml round bottom fraction collection tubes with caps</td><td>Preference of researcher</td><td></td><td></td></tr><tr><td>Need approximately 100 tubes</td><td></td><td></td><td></td></tr><tr><td>Fraction collector</td><td>GE Healthcare Life Sciences</td><td>Model: Frac-920, Product code: 18-1177-40</td><td>Alternate can be used.</td></tr><tr><td>Microscope slides, cover glass</td><td>Preference of researcher</td><td></td><td></td></tr><tr><td>Light/Fluorescent Microscope</td><td>Preference of researcher</td><td></td><td></td></tr></tbody></table>

separation of spermatogenic cell types using sta-put velocity sedimentation

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The ideal result from the STA-PUT procedure is a fairly noticeable separation of cells from the testes based on cell size and density. While cells isolated from the testes are sedimenting through the BSA gradient, several distinct bands of cells can be observed. Any clumps of cells tend to sink to the bottom of the gradient and will not contaminate the other fractions. A little further up the gradient will be the large somatic and meiotic cells. Farther up the gradient still will be smaller round spermatids. At the ...

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

The overall goal of this procedure is to separate the different types of cells found in the testes. First, harvest a mixed population of cells from the mouse. Testes load the cells and BSA gradient into the stay put apparatus.Now allow the cells to sediment through the BSA gradient. Then collect the fractions and combine them based on cell composition. Ultimately, velocity sedimentation with the stay put is used to separate different types of cells from the testes based on differences in size and density.The main advantage of this technique over existing methods like facts, is that one can isolate large amounts of different cell types with fairly high purity using simple glassware and gravity. Secure the two, two liter cylinders and the cell loading chamber to the top platform and connect all with two small pieces of tubing with tube clamps. Clamp all the tubes closed and seal the spout on the rightmost two liter cylinder.Place a small stir bar in the cell loading chamber and a larger stir bar in the leftmost two liter cylinder. Then place the two liter sedimentation chamber on the platform. Position the metal baffle directly on top of the opening in the bottom of the sedimentation chamber to prevent vortexing of the liquid and disruption of the cell gradient during fraction collection.Now put the lid on the sedimentation chamber. Apply a very small amount of vacuum grease to the ground glass joint of the three-way stop cock. Then clamp the stop cock to the bottom of the sedimentation chamber, connecting the ground glass joints of the stop cock and the sedimentation chamber.Next, connect the cell loading chamber to the right outlet of the stop cock with tubing. Close the stop cock, then attach the cell fractionation tubing to the left outlet of the stop cock and clamp the small tube at the very bottom. Pour the 2%BSA solution in the left two liter cylinder.Pour the 4%BSA solution in the right two liter cylinder. Make sure there are no large bubbles in the tubing that connects these cylinders. Pour Krebs buffer into the cell loading chamber and fill the tubing connecting this chamber with the sedimentation chamber.Verify that there are no large bubbles that might disturb the gradient. If necessary, squeeze or flick the tubing gently to remove the bubbles. Confirm that all of the crebs is in the tube and not in the cell chamber.Allow a small amount of Krebs buffer to flow into the sedimentation chamber. Then drain almost all the buffer into the cell fractionation tubing. In order to fill the tube and remove any large bubbles, place the fraction collector directly under the sedimentation chamber.Check that all the fraction tubes are in place and cover with plastic wrap to prevent contamination. Deencapsulate the testes in a separate plate containing eight milliliters of Krebs. Make an incision in the thin membrane that surrounds the seminiferous tubules with one pair of forceps.Hold the membrane now with another pair of forceps. Push the tubules out of and away from the membrane. Transfer four milliliters of Krebs containing the tubules to each conical tube containing 25 milliliters of collagenase solution.Shake at 33 degrees Celsius for 10 minutes until the tubules develop a spaghetti like appearance. Next, allow the tubules to settle for five minutes to the bottom of the tube. Pour out the supernatant and wash twice with 25 milliliters of Krebs at room temperature, allowing the tubules to settle to the bottom of the tube each time.Leave approximately five milliliters of Krebs in each tube. After incubating the tubules with trypsin. Use a wide bore pipette to agitate the solution containing the tubules.Pipetting them in and out approximately 10 times. The solution should begin to look more like a single cell suspension. Now filter each 30 milliliter single cell suspension through a 100 micron mesh cell strainer.After combining the cell suspensions, count the total number of cells. Make sure the stop cock is positioned to direct the flow from the cell chamber into the sedimentation chamber. Turn both stir bar plates onto a low setting.Then pour the cell suspension into the cell chamber. Open the stop cock for the cells to flow slowly into the sedimentation chamber at a rate of 10 milliliters per minute. Then close the stop cock to wash the cells out of the cell chamber.Pour five milliliters of 0.5%BSA solution and drain into the sedimentation chamber at a rate of 10 milliliters per minute. Close the stopcock and repeat the BSA wash four more times. Now open the clamps between the cell chamber and the two two liters cylinders to immediately drain the liquid into the sedimentation chamber at a rate of 40 milliliters per minute, adjust the flow rate so it takes approximately 20 to 30 minutes to load the BSA gradient into the sedimentation chamber.Monitor for a thin undisturbed layer of cells lying on top of the BSA gradient. Once most of the BSA is loaded, close the stop cock and turn it to the position that will allow liquid to drain from the sedimentation chamber into the fractionation tube. Now turn off the stir plates and allow the cells to sediment for one hour and 45 minutes.After sedimentation is complete, attach the fraction tube to the fraction collector. Adjust the flow rate with the stop cock so that 10 milliliters per collection tube is collected every 45 seconds. Once all fractions are collected microscopically analyze the fractions for different cell populations.Different cell types can be distinguished based on cell size and nuclear morphology to determine the purity of each fraction. Transfer 10 microliters of DPI stained cells onto a slide. Place a cover slip and analyze with a fluorescence microscope.Pull the fractions that are similar in size and nuclear morphology to create populations of cells. A typical preparation of approximately 22 testes. With this stay put procedure yields approximately 10 to the eighth cells.Perper metagenic cell type fractions can be analyzed quickly using a combination of light and fluorescent microscopy. Myotic, spermatogonia and somatic diploid cells are the largest cells found in the testes and will contain large nuclei that stain relatively homogenously with dpi. Round spermatids are smaller cells with smaller round nuclei, generally with a brightly staining chromo center condensing elongating spermatids are small cells that have sickle shaped nuclei.Once cell fractions are combined, purity can be further determined by western blood analysis of the cell lysates. Common markers of myotic cells are the Synap Neal Complex one proteins. Common markers of condensing spermatids are transition proteins or protamine.Once mastered, this technique can be done in seven hours if it's performed properly after its development. This technique paved the way for researchers in the field of spermatogenesis to explore cellular processes in different cell types from the testes.

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Research

JoVE Journal

Biology

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Learn STA-PUT velocity sedimentation to separate spermatogenic cell types from mouse testes based on size and density. This method enables isolation of large quantities of pure cell populations using simple glassware and gravity for reproductive biology research.

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

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