The authors would like to thank Guido Verhoeven and Ludo Deboel, Leuven, who were extremely helpful in establishing the isolation of primary Sertoli cells in the Meinhardt lab in Giessen. Monika Fijak, Gie&#223;en, is acknowledged for her help with figure 1A and advice. Studies were supported by the Deutsche Forschungsgemeinschaft through grant BH 93/1-1 (S.B.) and funding of the International Research Graduate College JLU Gie&#223;en (Germany)/Monash University (Melbourne, Australia) GRK 1871 (S.B.). Support was also obtained from a grant of the State of Hessen within the program &#34;Landes-Offensive zur Entwicklung Wissenschaftlich-&#246;konomischer Exzellenz&#34; (LOEWE) called &#34;M&#228;nnliche Infertilit&#228;t bei Infektion &#38; Entz&#252;ndung&#34; (MIBIE).

1. Animal Ethics Statement
Experiments described here were performed according to the guidelines of the Regierungspr&#228;sidium Giessen, Germany, as the local authority and confirm to the German Code of Practice for the Care and Use of Animals for Experimental Purposes (Permission no: GI 20/23 Nr. A 31/2012).
2. Preparation of Media, Enzyme Solutions and Animals
<ol>
	<li>Prepare 1% iodine alcohol by dissolving 1 g of iodine in 100 ml ethanol.</li>
	<li>Prepare 2x 500 ml Dulbecco&#180;s phosphate buffered saline (PBS) without Ca2+ and Mg2+ supplemented each with 7.5 ml D-glucose (100 g/L) and 5 ml 100x Penicillin/Streptomycin stock solution (15 mg/ml D-glucose, 50 U/ml Penicillin and 50 &#181;g/ml Streptomycin final).</li>
	<li>Supplement 1x 500 ml Roswell Park Memorial Institute (RPMI) 1640 medium with 5 ml 100x Penicillin/Streptomycin stock solution (50 U/ml Penicillin and 50 &#181;g/ml Streptomycin final).</li>
	<li>Prepare a Trypsin-DNase I solution (2.5 mg/ml Trypsin and 10 &#181;g/ml DNase I) by adding 25 mg Trypsin and 0.1 ml DNase I (1 mg/ml DNase I) to 9.9 ml PBS.</li>
	<li>Dissolve 50 mg Trypsin inhibitor in 5 ml PBS for Trypsin inhibitor A solution (10 mg/ml). Dissolve 25 mg Trypsin inhibitor in 10 ml PBS for Trypsin inhibitor B solution (2.5 mg/ml).</li>
	<li>For a Collagenase-Hyaluronidase-Deoxyribonuclease I (DNase I) solution dissolve 10 mg Collagenase A (1 mg/ml final), 10 mg Hyaluronidase (1 mg/ml final) and 0.1 ml DNase I (1 mg/ml DNase I) (10 &#181;g/ml final) in 10 ml PBS.</li>
	<li>Prepare a Hyaluronidase-DNase I solution by adding 10 mg Hyaluronidase (1 mg/ml final) and 0.1 ml DNase I solution (1 mg/ml) (10 &#181;g/ml final) to 9.9 ml PBS.</li>
	<li>Order Wistar rats on time so that they are 19 days old on the day of Sertoli cell isolation. 
	Note: The described protocol is designed for 10 rats but can be modified for a minimum of 5 and a maximum of 20 rats. The volumes of all solutions have to be adjusted accordingly.</li>
	<li>Prepare Oil Red O stock solution by dissolving 0.35 g Oil Red O in 100 ml isopropanol. Stir O/N, filter through 0.2 &#181;m and store at RT for up to one year.</li>
	<li>Prepare Oil Red O staining solution by mixing 6 ml Oil Red O stock solution with 4 ml water (3:2). After 10-20 min filter through 0.2 &#181;m. Use within the next 2 hr.</li>
	<li>Prepare a 4% (w/v) formaldehyde solution in a ventilated hood by adding 4 g paraformaldehyde powder (PFA) to 80 ml of 1x PBS while stirring gently. Heat to ~60 &#176;C, add 1 ml of 1 M NaOH and continue stirring until the paraformaldehyde is dissolved. Let the solution cool down to RT, adjust the pH to 7.4 with 1 M HCl and the volume to 100 ml with 1x PBS. Filter the solution (0.45 &#181;m) and use fresh or store in aliquots at &#8722;20 &#176;C for several months. 
	Note: Polymerized formaldehyde (PFA) depolymerizes to monomeric formaldehyde in water. This process is catalyzed by moderate heating and a slightly alkaline pH. Prepare all enzyme solutions freshly and filter sterilize (0.2 &#181;M) them on the day of use. If a different manufacturer for an enzyme (see Materials/Equipment Table) is used, carefully adjust its concentration/activity. 
	In the following protocol a &#34;pipette&#34; stands for a serological pipette.</li>
</ol>
3. Preparation of Seminiferous Tubules
<ol>
	<li>Anesthetize 10 male Wistar rats one by one in a desiccator using CO2 and a flow rate that displaces at least 20% of the chamber volume per minute. Wait until breathing stops, test anesthesia by squeezing a foot pad, and if there is no reaction euthanize each rat by cervical dislocation. Drain the blood by sectioning jugular vein and carotid artery (vagina carotica) under flowing water. Disinfect the abdomen by wiping with 70% ethanol.</li>
	<li>Using forceps lift the skin from the abdominal muscles and cut out an oval shaped skin lobe that is extending from the pubic symphysis to the sternum (Figure 2A) and flap it upwards onto the chest. Next, grab the abdominal muscles and make a medial incision from the pubic symphysis to the sternum.
	<ol>
		<li>Squeeze the abdomen with the thumbs from pelvis upwards in order to push the testes out of the lower pelvis. Pick the epididymal fat pad (Figure 2B) with forceps for pulling up the testis further. Cut the spermatic cord (Figure 2B), leaving the tunica albuginea intact, and collect all testes in 20 ml PBS in a 50 ml conical tube (Figure 2C).</li>
	</ol>
	</li>
	<li>After collecting all testes, disinfect them by adding an equal volume (20 ml) of 1% (w/v) iodine in ethanol. Invert the tube twice and decant the supernatant immediately. Quickly wash the testes twice with 25 ml PBS each (Figure 2D).</li>
	<li>Transfer the testes to a Petri dish containing 15 ml PBS (Figure 2E). Grasp each testis firmly by forceps at one end, make a small incision into the tunica albuginea at the opposite end, and squeeze out the tubules using closed scissor blades as a scraping tool. 
	Note: The tubules should still form a compact testicle-shaped bundle with only a few tubules extending into the solution in order to enable homogeneous enzymatic digestion (Figure 2F).</li>
	<li>Transfer the de-capsulated testes to a 100 ml screw-cap bottle containing 10 ml Trypsin-DNase I solution. Perform the digestion in a shaking water bath at 32 &#176;C (120 oscillations/min). After 4 min evaluate the tubules with the naked eye for dispersion of tubules. Once tubules start to disperse into the solution, stop the digestion immediately. If necessary, continue incubation for up to 2 min.</li>
	<li>Stop trypsin digestion by adding 5 ml of trypsin inhibitor solution A. Thoroughly mix the solution by pipetting up and down 3-4 times using a 10 ml pipette and transfer it to a 50 ml conical tube.</li>
	<li>After 5 min of incubation observe the tubules settle by unit gravity, carefully remove the supernatant by using the 10 ml pipette from step 3.6 and add 10 ml of trypsin inhibitor B solution in order to fully stop trypsin digestion. Resuspend the tubules by pipetting up and down 2-3 times.</li>
	<li>In order to remove contaminating interstitial cells wash the tubules 9 times with 25 ml PBS each. Resuspend tubules in each wash by using a 25 ml pipette and allow them to settle by unit gravity for 10 min. Always use the same 25 ml pipette for pipetting PBS, resuspension and removal of the supernatant.</li>
</ol>
4. Removal/Isolation of Peritubular Cells (PTCs)
<ol>
	<li>Transfer all tubules to a 100 ml screw-cap bottle and add the Collagenase-Hyaluronidase-DNase I solution (Figure 3A). Perform digestion for 10 min in a shaking water bath at 32 &#176;C (120 oscillations/min).</li>
	<li>Pipet a drop of the suspension on a glass slide and quickly check the tubules under an inverted light microscope for routine cell culture at 100x magnification. If digestion is not advanced enough continue incubation for additional 2 min (do not count the time needed for the microscopical check). 
	Note: During this step all tubules get shortened in length and tubule edges should become rough (Figure 3B and C). Rough edges are indicative for the release of peritubular cells.</li>
	<li>Transfer the suspension with digested tubules to a 50 ml conical tube using the 25 ml pipette from step 3.8. Wash the 100 ml bottle with 10 ml PBS and add it to the tubules in the conical tube. Allow the digested tubules to settle by unit gravity for 10 min, and carefully aspirate the supernatant, containing the PTCs, using the 25 ml pipette again, and transfer it to a new conical tube. For aspiration of the last few milliliters use a 5 ml pipette in order to prevent any carry-over of tubules.</li>
	<li>Add 20 ml RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) to the collected tubule supernatants, mix and centrifuge at 300 x g for 10 min at RT without using the break.</li>
	<li>Carefully decant the supernatant and resuspend the pelleted PTCs in 20 ml RPMI 1640 medium supplemented with 10% heat-inactivated FBS and seed 4 ml each on five T75 flasks containing 16 ml medium each. Incubate at 37 &#176;C in 5% CO2 atmosphere (Figure 4A).</li>
	<li>On the 3rd day of culture trypsinize the PTCs and split them 1:2 on T75 flasks containing 16 ml RPMI 1640 medium supplemented with 10% heat-inactivated FBS each (yields 10 flasks altogether) (Figure 4B). Continue incubation at 37 &#176;C in 5% CO2 atmosphere.</li>
	<li>On the 5th day of culture trypsinize the PTCs again and plate them on 6-well or 24-well plates depending on the experiment (one T75 flask per plate). Experiments can be performed on day 6.</li>
</ol>
5. Isolation of Sertoli Cells (SCs)
<ol>
	<li>Resuspend the settled tubules from step 4.3 thoroughly in 25 ml of PBS by using a 25 ml pipette and allow them to settle by unit gravity for 12 min. Use the same 25 ml pipette for pipetting PBS, resuspension of tubules and removal of supernatants. Repeat the wash 3 times (4 washes in total).</li>
	<li>After the last wash transfer the digested tubules to another 100 ml screw-cap bottle containing the Hyaluronidase-DNase I solution. Perform the digestion in a shaking water bath at 32 &#176;C (120 oscillations/min).</li>
	<li>After 5 min check the tubules again by light microscopic inspection at 100X magnification as described in step 4.2. 
	Note: Short tubules, tubular aggregates and released cells should be visible (Figure 3D).
	<ol>
		<li>Allow the tubular aggregates settle for 10 min, and aspirate the supernatant using a 25 ml pipette. Wash aggregates 4 times using 25 ml PBS and a sedimentation time of 10 min for each washing step (Figure 3E).</li>
	</ol>
	</li>
	<li>Add 20 ml of RPMI 1640 medium and pass the suspension 10 times through an 18 G needle mounted on a 20 ml syringe. Avoid air bubbles. 
	Note: Pulling the suspension in and out through the needle is considered as one pass. The disruption and homogenization of cells by hydrodynamic shearing is a standard technique in the preparation of cells that tend to aggregate. The passing of cells through a hypodermic needle of small inner diameter is unlikely to have an impact on their functional properties25. Figure 5C and D shows the normal ultrastructure of purified Sertoli cells on day 4 of culture.
	<ol>
		<li>Pipet a drop of the suspension on a glass slide and quickly check the tubules under an inverted light microscope for routine cell culture at 10X magnification if the aggregates are all dispersed (Figure 3F). Filter the cell suspension through a 70 &#181;m cell strainer in order to obtain a pure single cell suspension in the filtrate.</li>
	</ol>
	</li>
	<li>Centrifuge the filtrate of step 5.4 at 200 x g for 10 min at RT without using the break. Carefully aspirate the supernatant using a 25 ml pipette, and resuspend the Sertoli cells in 40 ml RPMI 1640 medium (serum-free).</li>
	<li>Mix 100 &#181;l cell suspension with 100 &#181;l of Trypan Blue Stain and count cells with a Neubauer Improved chamber. Adjust the cell concentration to 3 x 106 cells/ml according to the counting result. Seed 1 ml per well on a 6-well plate (=day 1). If an Oil Red O staining (step 6) is planned put a cover slip into one or a few wells before seeding Sertoli cells. 
	Note: Sertoli cells in suspension settle quickly so that the tube should be briefly shaken every time before a cell aliquot is taken with the pipette. Until day 4 they firmly attach to the substratum.</li>
	<li>In order to remove floating or adherent germ cells on day 4 (Figure 4C) wash each well of the 6-well plates used for plating isolated Sertoli cells (step 5.6) with PBS (Figure 4D). Aspirate the medium, add 1-2 ml of PBS to each well and shake the 6-well plates horizontally on a table vigorously but without spilling PBS. Repeat the wash 3 times and perform the same washing procedure on day 5 and 6 (Figure 4E). 
	Note: On day 4 Sertoli cells have firmly attached and show a cobblestone like pattern under the inverted light microscope (Figure 4C). Experiments can be performed from day 7 onwards.
	<ol>
		<li>(Alternative for step 5.7) For the removal of floating and adherent germ cells perform a hypotonic shock 3 days after seeding on 6-well plates. Remove the medium and add 1 ml of 20 mM Tris pH 7.5 taken directly out of the fridge. Aspirate the Tris buffer after 90 to 120 sec but not later. Wash cells twice with PBS, and add 2 ml of RPMI 1640 medium (serum-free). Optionally, perform experiments the next day. 
		Note: The longer the duration of the hypotonic shock is the more germ cells can be removed but the more Sertoli cells get lost as well. The first PBS wash should be performed quickly but with care in order not to displace any Sertoli cells. Directly after hypotonic treatment numerous vacuoles of different sizes can be observed in the cytoplasm by phase contrast microscopy. Vacuoles disappear within a few hours after hypotonic treatment.</li>
	</ol>
	</li>
</ol>
6. Oil Red O Staining
<ol>
	<li>Perform all steps at RT. On day 7 wash Sertoli cells grown on a cover slip (step 5.6) with PBS twice and fix them with 10% formalin in PBS. After 10 min replace formalin with fresh solution, and continue fixation for another 60 min. 
	Note: All solutions are added to the well(s) with a cover slip and aspirated before the next step.</li>
	<li>Aspirate formalin, wash cells with water twice and add 60% isopropanol. After 5 min allow cells to air dry for a few minutes.</li>
	<li>Add 1 ml Oil Red O staining solution per well and incubate for 10 min. Aspirate the solution, and wash cells immediately 4 times with water and mount cover slips in glycerol-PBS (9:1). Take bright field images with a routine light microscope (Figure 4F).</li>
</ol>
7. Immunofluorescence Staining
<ol>
	<li>Wash PTCs (from step 4.5) or Sertoli cells (from step 5.5) grown on a cover slip with PBS twice and fix them with 4% PFA for 10 min at RT.</li>
	<li>Incubate with 5% BSA in PBS for 1 hr at RT. Discard the BSA-PBS solution and add fresh BSA-PBS solution containing actin (smooth muscle) (for PTCs) or vimentin (for Sertoli cells) primary antibody (dilution 1:100 for both antibodies) and incubate at 4 &#176;C O/N. 
	Note: Incubation with BSA blocks non-specific binding of the primary antibody.</li>
	<li>Wash cover slips 3 times for&#160; 5 min in PBS-0.1% Tween 20 and incubate with green-fluorescent dye conjugated goat anti-mouse secondary antibody (dilution 1:1,000) at RT for 1 hr in the dark.</li>
	<li>Wash cover slips 3 times for 5 min in PBS-0.1% Tween 20 and mount them in DAPI mounting medium.</li>
</ol>
8. Ultrastructural Analysis
<ol>
	<li>Wash Sertoli cells (from step 5.6) grown on a 6 cm cell culture plate with PBS and fix them for 2 hr&#160;at 4 &#176;C in a mixture of 2.5% glutaraldehyde, 2.5% paraformaldehyde and 0.05% picric acid in 67 mM cacodylate buffer (pH 7.4) according to Ito and Karnovsky26.</li>
	<li>Perform post-fixation in 1% osmium tetroxide followed by an O/N incubation in 0.3% uranyl acetate dissolved in 50 mM maleate buffer (pH 5). Embed samples in embedding media according to standard procedures. Cut thin sections, contrast them with lead citrate and examine with an electron microscope.</li>
</ol>

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+passing+mesenchymal+stem+cells+through+smaller+bore+size+needles+for+subsequent+use+in+patients+for+clinical+or+cosmetic+indications.">Impact of passing mesenchymal stem cells through smaller bore size needles for subsequent use in patients for clinical or cosmetic indications.</a> J. Transl. Med. 10, 229 (2012).</li><li>Ito, S., Karnovsky, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde-glutaraldehyde+fixatives+containing+trinitro+compounds.">Formaldehyde-glutaraldehyde fixatives containing trinitro compounds.</a> J. Cell. Biol. 39, 168-169 (1968).</li><li>Galdieri, M., Ziparo, E., Palombi, F., Russo, M. A., Stefanini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pure+Sertoli+cell+cultures:+a+new+model+for+the+study+of+somatic-germ+cell+interactions.">Pure Sertoli cell cultures: a new model for the study of somatic-germ cell interactions.</a> J. Androl. 5, 249-254 (1981).</li><li>Fijak, M., Meinhardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+testis+in+immune+privilege.">The testis in immune privilege.</a> Immunol. Rev. 213, 66-81 (2006).</li><li>Jegou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sertoli+cell+in+vivo+and+in+vitro.">The Sertoli cell in vivo and in vitro.</a> Cell Biol. Toxicol. 8, 49-54 (1992).</li><li>Pineau, C., Le Magueresse, B., Courtens, J. L., Jegou, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+in+vitro+the+phagocytic+function+of+Sertoli+cells+in+the+rat.">Study in vitro the phagocytic function of Sertoli cells in the rat.</a> Cell Tissue Res. 264, 589-598 (1991).</li><li>Ren, Y., Savill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis:+the+importance+of+being+eaten.">Apoptosis: the importance of being eaten.</a> Cell Death Differ. 5, 563-568 (1998).</li><li>Ducray, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+mouse+Sertoli+cell+line+producing+rat+androgen-binding+protein+(ABP).">Establishment of a mouse Sertoli cell line producing rat androgen-binding protein (ABP).</a> Steroids. 63, 285-287 (1998).</li><li>Konrad, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+Sertoli+cells+express+epithelial+but+also+mesenchymal+genes+after+immortalization+with+SV40.">Rat Sertoli cells express epithelial but also mesenchymal genes after immortalization with SV40.</a> Biochim. Biophys. Acta. 1722, 6-14 (2005).</li></ol>

The authors have nothing to disclose.

The seminiferous tubules are bounded by a circular layer of peritubular cells and a basal lamina on the luminal side. Sertoli cells are resting on the basal lamina, establish the blood testis barrier through formation of occluding junctions between adjacent cells and provide the structural framework for the organisation of the seminiferous epithelium. Sertoli cells and adjacent spermatogenic cells maintain intimate contacts throughout germ cell development providing physical contact and communication alike. Therefore, wh...

Testes produce male gametes and sexual hormones, i.e., androgens. The organ is composed of two compartments. In the interstitial compartment, that represents about 10-12% of total testicular volume1, steroidogenesis takes place within the Leydig cells. The tubular compartment represents about 60-80% of the testicular volume1 and contains germ cells and two types of somatic cells - peritubular cells and Sertoli cells. The testis is divided by connective tissue septa into 250-300 lobules, each containing 1-3 highly convoluted seminiferous tubules. These tubules are enclosed by a basal membrane, a sheet of collagen, and a circumferential layer of peritubular cells (Figure 1A).
The germinal epithelium is located on the luminal side of the basal membrane. Sertoli cells are large elongated cells that span the whole germinal epithelium from the basal membrane to the lumen. They are strongly attached to the basal membrane and form a continuous cellular sheet through basolateral organized tight junctions that occludes the germinal epithelium from the interstitium and represents the blood-testis barrier. Sertoli cells have prominent cytoplasmic projections and ramifications that enable them to get into tight morphological and functional contact with a species-specific but constant number of germ cells. Diploid germinal stem cells proliferate and differentiate into spermatogonia.
During meiosis I short-lived tetraploid spermatocytes are generated that develop further into four haploid spermatids during meiosis II. All germ cells are interconnected by cytoplasmic bridges so that they form a cellular net. The principal event during maturation of spermatids is the extrusion of large parts of the cytoplasm, forming residual bodies, in a process called spermiogenesis. Residual bodies are phagocytosed by Sertoli cells. Late spermatids are then released into the tubular lumen and transported into the epididymis for further maturation. Sertoli cells and germ cells appear to mutually coordinate spermatogenesis topographically and functionally.
Preparation of individual testicular cell types started almost a century ago when small testicular pieces were cultivated and cell types identified by microscopy2. By careful dissection of the tubules after opening the tunica albuginea using fine-tipped forceps it was later possible to separate tubular and interstitial compartments3. In 1975 Welsh and Wiebe introduced a collagenase treatment in order to free the tubules from adhering interstitial tissue and a pancreatin treatment for removal of the outer peritubular cell layer4. From early on young immature rats (around 20 days old) had been used in which the Sertoli cells comprise a large fraction of the tubular cell population because spermatogenesis has not begun yet. At this age rat Sertoli cells cease to divide, and tight junctions between neighboring cells form so that the blood-testis barrier is established5.
Independent of Welsh and Wiebe Dorrington et al. introduced a combination of trypsin and deoxyribonuclease followed by soybeen trypsin inhibitor and collagenase treatment that was published in the same year6. Both groups also used mechanical force (drawing the digested tubular fragments repeatedly through a syringe needle or a Pasteur pipette respectively) in order to produce a homogeneous cell suspension for plating that contains approximately 70% Sertoli cells. After 3 days in culture, using a medium that is serum-free, the percentage of Sertoli cells increases to approximately 90%. This could be largely attributed to the death of contaminating germ cells. Residual peritubular cells (PTCs), however, stay firmly attached via their extracellular matrix. PTCs, but not Sertoli cells, are known to produce fibronectin that can serve as a marker protein for estimating contamination with PTCs. Therefore, Tung et al. reasoned that an additional hyaluronidase treatment might improve the purity of the Sertoli cell fraction7. Indeed they could show that an additional treatment was able to reduce contamination by PTCs approximately 20-fold, which becomes particularly evident when in comparison a serum-containing medium is used for cultivating purified Sertoli cells. From then on the improved procedure of Tung et al. became the prevailing protocol and was extensively used by other major groups in the field8 (Figure 1B).
During collagenase treatment the majority of PTCs are released and can be isolated in parallel to Sertoli cells. Whereas the PTCs vigorously proliferate and do not respond to follicle-stimulating hormone (FSH), Sertoli cells do not undergo mitosis anymore and respond to FSH by characteristic morphological changes and an increase in cyclic adenosine monophosphate (cAMP) concentration9. Very similar enzymatic digestion protocols can be used for the isolation of primary Sertoli cells from other animals like man10, mouse11,12, Siberian hamster13 or yak14. For the removal of large amounts of contaminating germ cells a hypotonic shock can be employed at the end of the isolation procedure15. This allows also the efficient isolation of Sertoli cells from adult rat testes16. An enriched Sertoli cell suspension can also be separated from germ cells by plating the suspension on lectin dishes coated with Datura Stramonium agglutinin17. Only Sertoli cells and a few residual PTCs adhere to the lectin plates.
Primary Sertoli cell cultures, mainly from the rat, have been used initially for investigating responsiveness to hormones or establishing cell lines like the mouse Sertoli cell line TM418. This cell line has been investigated in more than a hundred studies until today. In a translational approach Sertoli cells have been utilized for immuno-protection of co-cultured cells and tissues like in the co-transplantation of xeno- or allogeneic pancreatic islets for long-term graft survival without systemic immunosuppression19. Isolated Sertoli cells have been also used in co-culture experiments for studying epithelial-mesenchymal (Sertoli cells - PTCc) and somatic-germ cell (Sertoli cells - germ cells) interactions20,21. Recently primary Sertoli cells have been employed for investigating the expression of Toll-like receptors and the secretion of proinflammatory cytokines as well as the signal transduction cascades leading to cytokine expression following infection with nonpathogenic and uropathogenic E. coli22. Other recent investigations were using Sertoli cells for studying testicular immune privilege23 and demonstrated that testosterone pre-treatment suppresses the LPS-induced inflammatory response24.

<table border="0" cellpadding="0" cellspacing="0" width="813">
	<tbody>
		<tr height="80">
			<td height="80" style="height:80px;width:289px;">Actin (smooth muscle) antibody clone 1A4</td>
			<td style="width:111px;">Dako</td>
			<td style="width:124px;">M0851</td>
			<td style="width:289px;">Monoclonal mouse anti human antibody. 
			Use 1:100 dilution for immunofluorescence.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Albumin bovine fraction V 
			Standard grade, lyophilized</td>
			<td style="width:111px;">Serva</td>
			<td style="width:124px;">11930.04</td>
			<td style="width:289px;">Filter (0.45 &#181;m) BSA solutions used for immunofluorescence.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Corning&#8482; Cell Strainers 70&#160;&#181;m</td>
			<td style="width:111px;">Corning</td>
			<td style="width:124px;">431751</td>
			<td>White color</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Collagenase A from Clostridium histolyticum</td>
			<td style="width:111px;">Roche</td>
			<td style="width:124px;">10103586001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">DAPI mountant ProLong&#174; Gold Antifade</td>
			<td style="width:111px;">Life technologies</td>
			<td style="width:124px;">P-36931</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">D-glucose</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">G8644</td>
			<td>100 g/l</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Dulbecco&#180;s PBS without Ca2+/Mg2+</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">DNase I</td>
			<td style="width:111px;">Roche</td>
			<td style="width:124px;">10104159001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Electron microscope</td>
			<td style="width:111px;">Zeiss</td>
			<td>EM 109S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Fluorescence microscope</td>
			<td style="width:111px;">Zeiss</td>
			<td style="width:124px;">Axioplan 2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Hyaluronidase from bovine testis</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">H3506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Inverted light microscope</td>
			<td style="width:111px;">Olympus</td>
			<td style="width:124px;">CKX41</td>
			<td>Routine cell culture microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Mouse IgG / IgM (H+L) polyclonal 
			secondary Antibody&#160;</td>
			<td style="width:111px;">Life technologies</td>
			<td style="width:124px;">A-10684</td>
			<td>Alexa Fluor&#174; 488 conjugate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Oil Red O</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">O0625</td>
			<td style="width:289px;">Has replaced Sudan III and Sudan IV 
			because of brighter color.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Paraformaldehyde</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">P6148</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:289px;">Penicillin (5,000&#160;U/ml)/ 
			Streptomycin (5,000 &#181;g/ml)</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">15070-063</td>
			<td>100x solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">RPMI-1640</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">21875-034</td>
			<td>Contains 300 mg/L L-glutamine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Trypsin from porcine pancreas</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">T5266</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Trypan Blue Stain (0.4%)</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:124px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:289px;">Trypsin inhibitor from soybean</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">T6522</td>
			<td style="width:289px;">The Sigma product is considerably cheaper than the previously used 
			BPTI (Aprotinin) from Roche.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:289px;">Vimentin antibody (V9)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:124px;">V6630</td>
			<td style="width:289px;">Monoclonal mouse anti pig vimentin antibody.&#160; Use 1:100 dilution for immunofluorescence.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:289px;">Wistar WU rats</td>
			<td style="width:111px;">Charles River</td>
			<td style="width:124px;">N/A</td>
			<td>Should be 19 days old on day of experiment.</td>
		</tr>
	</tbody>
</table>

isolation of sertoli cells and peritubular cells from rat testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

The described procedure allows isolation of approximately 12 x 107 Sertoli cells from 10 rat testes. 3 x 106 cells are plated per well on a 6-well plate so that six to seven 6-well plates are available for experiments on day 7. The Trypsin-DNase I digestion is the most critical step during Sertoli cell isolation. If digestion at this point is advancing too far, the ratio of germ cells/Sertoli cells will disproportionally increase up to the end. During day 3-6 of cult...

Watch this Scientific Journal Video about Isolation of Sertoli Cells and Peritubular Cells from Rat Testes at JoVE.com

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

isolation-of-sertoli-cells-and-peritubular-cells-from-rat-testes

Research

JoVE Journal

Immunology and Infection

22.3K Views.  Justus-Liebig-Universität Gießen. Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

Video: Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...