Diese Arbeit wird von NIH PBS (R21 AI095484) unterstützt. Wir danken NHI Tetramer Core Facility für CD1d Tetramer.

1. Vorbereitung der Instrumente, Kultur, Medien und Reagenzien <ol><li> Instrumente: Bereiten Sie die Reagenzien unter sterilen Bedingungen und unter Verwendung einer Steril. </li><li> Herstellung von 1 l RPMI-Gewebekulturmedium mit 5% fötalem Rinderserum, 2,1 g Natriumbicarbonat, 10 ml Glutamat 100x, 10 mg HEPES, 1 mg Natriumpyruvat und 10 ml 100x Penicillin / Streptomycin. Hinweis: Die empfohlene Gewebekulturmedium RPMI, ist austauschbar mit DMEM. </li><li> Vorbereitung 5% Collagenase &quot;D&quot;-Lösung (5 ml Collagenase &quot;D&quot; zu 9 ml Gewebekulturmedium). </li><li> Machen Percoll-Lösung bei drei verschiedenen Konzentrationen: 100%, 80% und 40%. Die folgenden Konzentrationen sind 1 Probe berechnet. Halten Lösungen bei Raumtemperatur. <ol><li> Percoll Lösung: 9 ml unverdünntem Percoll (aus Stammlösung); 1 ml PBS 10x. </li><li> Percoll-Lösung: 8 ml Percoll 100%; 2 ml PBS 1x. </li><li> Percoll-Lösung:4 ml Percoll 80%; 4 ml PBS 1x. </li></ol></li><li> Auffüllen auf 1 L der Fluoreszenz-aktivierten Zellsortierung (FACS)-Puffer; 5 ml 10% BSA (0,1% BSA), 1 ml 10% Natriumazid (0,1%), 2 ml EDTA-Agent, machen bis zu 1.000 ml mit 1x PBS. </li></ol> 2. Vorbereitung von Nieren Verdauung <ol><li> Nach dem Erwerb aller erforderlichen institutionellen Genehmigung opfern und exsanguinate die Mäuse mit Standardmethoden und nach den aktuellen Vorschriften und Tierpflege-Richtlinien. <ol><li> Betäuben die Maus mit Pentobarbital (0,07 mg / kg). </li><li> Fahren Sie mit dem Ausbluten. </li><li> Platzieren Sie die Maus in Rückenlage auf einem Operationstisch. </li><li> Sprühen Sie den Bauchbereich mit 70% Ethanol. </li></ol></li><li> 10 ml Collagenase D Lösung der Petrischale. </li><li> Entfernen und entkapseln beide Nieren aus jeder Maus. <ol><li> Öffnen abdomimal Hohlraum, indem eine Mittelschnitt durch die Bauchhaut und Peritoneum vom Brustbein bisdas Schambein. </li><li> Die linke Niere freizulegen, indem der Darm seitlich zur Seite. </li><li> Trennen Sie vorsichtig die linke Kapsel mit der Zange. Hinweis: Die Kapsel wird als transparente Schicht rund um die Nieren-und teilweise durch perinephric Fettgewebe bedeckt. </li><li> Entfernen Sie die linke Niere durch Schneiden der Stiel Schiffe, die mit einer chirurgischen Schere. </li><li> Wiederholen Sie diesen Vorgang für die rechte Niere. Hinweis: Die rechte Niere ist Schädel und näher an der Mittellinie (Blütenstiele sind kürzer). </li></ol></li><li> Legen Sie die Niere in der Petrischale. </li><li> Schneiden Sie das Nierengewebe in kleine Stücke (1-2 mm in der Abmessung) mit einem normalen Metallformung Klinge und legen die Stücke in den Collagenase-Lösung für 30-45 min im Brutschrank bei 37 ° C (Abbildung 1). </li></ol> 3. Vorbereitung von Nieren mononukleären Zellen (KMNC) aus der Niere Verdauung <ol><li> Eine Zellsuspension zu erhalten, der NiereVerdauung durch mechanisches Aufbrechen der Gewebe mit einem Sieb (70 um) und Resuspension in 25 ml Gewebekulturmedium und mischen. </li><li> Zentrifuge bei 400 × g für 10 min bei 4 ° C, um die Zellsuspension pelletieren. </li><li> Re-suspend das Pellet in 4 ml 40% Percoll-Lösung und sanft überlagern auf 4 ml 80% Percoll-Lösung mit einer Übertragungsplastikpipette, sehr langsam und vorsichtig. Dies wird in zwei Phasen klar durch eine transluzente Schicht getrennt führen. Auf der Oberseite wird eine gelbe Schicht, die den Lipiden sein. </li><li> Zentrifugieren bei 1500 × g für 30 min bei Raumtemperatur mit der Zentrifuge in Brems Aus-Modus. </li><li> Durch Absaugen entfernen das gelbe und dicke Deckschicht (ca. 1-2 ml). </li><li> Sammeln nur die leichte weißlich durchscheinende Schicht von der Grenzfläche der beiden Phasen mit einer Transferpipette. Diese Suspension sehr sorgfältig übertragen in eine 15 ml konischen Röhrchen mit 2 ml Gewebekulturmedium und fügen Sie dann 13 ml Medium, um ein Volumen von insgesamt machen15 ml. </li><li> Zentrifuge bei 400 × g für 10 min bei 4 ° C. </li><li> Resuspendieren der Proben in 1 ml des Mediums. </li><li> Zähle die Anzahl der KMNC mit Trypanblau-Ausschluss auf einer Zählkammer und das Ergebnis wird als Zahl der KMNC pro ml pro Niere. </li><li> Einmal wie oben waschen. </li></ol> 4. Isolierung von hämatopoetischen (CD45 +) Zellen aus KMNC (Schritt I) <ol><li> Resuspendieren der Zellen bis zu 10 7 in 90 ul des Laufpuffers. </li><li> In 10 ul CD45-Mikroperlen und Inkubation für 15 min bei 4 ° C </li><li> 1 ml Laufpuffer, um die Reaktion zu stoppen. Zentrifuge bei 400 × g für 10 min bei 4 ° C und Resuspendieren in 500 ul Laufpuffer. </li><li> Vorbereitung der Magnetspalte, indem an dem Magneten und gründlich mit 3 ml Laufpuffer. </li><li> Übergeben Sie die Zellsuspension durch die Magnetsäule und sammeln Sie die unmarkierte Zellen, die durch die Säule. </li><li> Mit 3 ml runn Waschen Sie die Zellen dreimalten Puffer. Dann sammeln das Abwasser. </li><li> Entfernen Sie die Spalte aus dem Separator und Pipette 5 ml Laufpuffer auf die Säulen und spülen Sie die markierten Bruch sofort. Diese Fraktion enthält die CD45 +-Zellen. Zählen Sie die Zellen. </li><li> Um die absolute Anzahl verschiedener T-Zellen in Teilmengen Nieren Errechnung der Gesamtzahl von KMNC durch den Prozentsatz positiver Zellen mittels Durchflusszytometrie bestimmt (Abbildung 2). </li></ol> 5. Isolierung von DN-T-Zellen aus CD45 + Vorbereitung durch negative Selektion (Schritt II) <ol><li> Add 2,5 ul (0,5 mg / ml, 1,25 &amp;mgr; g) von jedem der folgenden biotinylierten Antikörper: anti-CD4, anti-CD8, anti-Fc-Rezeptor (CD16/32), Anti-MHC-Klasse II (IA b), anti- CD1d Tetramer PBS-57 für alle 10 7 hämatopoetischen Zellen. </li><li> Inkubieren Zellen für 30 min in kalten Raum. </li><li> 1 ml anti-Biotin-Mikrokügelchen. </li><li> Inkubation für 30 min in einem kalten Raum. </li><li> Das Röhrchen wird in ter Magnet und fahren Sie mit Erschöpfung. </li><li> Zählen der lebensfähigen Zellen, die Gesamtzahl von DN-T-Zellen in der negativen Fraktion unter Verwendung einer Zählkammer bestimmen. </li><li> Überprüfen Reinheit der DN-T-Zellen mit Durchflusszytometrie, indem Sie dieses Gating-Strategie: erste Tor CD45 +, zweite Tor TCRαβ +. Ausschließen CD1d +-Zellen. Grundstück CD4 + vs CD8 + T-Zellen auf DN (Abbildung 3) zu identifizieren. </li><li> Multiplizieren Sie die Gesamtzahl der in der Reinheit bestimmt Prozentsatz. Hinweis: Die Verwendung dieses Protokolls zu einer Bevölkerung von mehr als 90% rein DN T-Zellen. </li></ol>

<ol>
	<li>Zhang, Z. X., Yang, L., Young, K. J., DuTemple, B., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+previously+unknown+antigen-specific+regulatory+T+cell+and+its+mechanism+of+suppression.">Identification of a previously unknown antigen-specific regulatory T cell and its mechanism of suppression.</a> Nat Med. 6, 782-789 (2000).</li><li>Ford, M. S., Young, K. J., Zhang, Z., Ohashi, P. S., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+regulatory+function+of+lymphoproliferative+double+negative+T+cells+in+vitro+and+in+vivo.">The immune regulatory function of lymphoproliferative double negative T cells in vitro and in vivo.</a> J Exp Med. 196, 261-267 (2002).</li><li>Ford McIntyre, M. S., Young, K. J., Gao, J., Joe, B., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+in+vivo+trogocytosis+as+a+mechanism+of+double+negative+regulatory+T+cell-mediated+antigen-specific+suppression.">Cutting edge: in vivo trogocytosis as a mechanism of double negative regulatory T cell-mediated antigen-specific suppression.</a> J Immunol. 181, 2271-2275 (2008).</li><li>Young, K. J., Yang, L., Phillips, M. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-lymphocyte+infusion+induces+transplantation+tolerance+by+activating+systemic+and+graft-infiltrating+double-negative+regulatory+T+cells.">Donor-lymphocyte infusion induces transplantation tolerance by activating systemic and graft-infiltrating double-negative regulatory T cells.</a> Blood. 100, 3408-3414 (2002).</li><li>Chen, W., Ford, M. S., Young, K. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+in+vitro-generated+DN+T+regulatory+cells+induces+permanent+cardiac+allograft+survival+in+mice.">Infusion of in vitro-generated DN T regulatory cells induces permanent cardiac allograft survival in mice.</a> Transplant Proc. 35, 2479-2480 (2003).</li><li>Young, K. J., DuTemple, B., Phillips, M. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+graft-versus-host+disease+by+double-negative+regulatory+T+cells.">Inhibition of graft-versus-host disease by double-negative regulatory T cells.</a> J Immunol. 171, 134-141 (2003).</li><li>Mohamood, A. S., 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=Gld+mutation+of+Fas+ligand+increases+the+frequency+and+up-regulates+cell+survival+genes+in+CD25+CD4++TR+cells.">Gld mutation of Fas ligand increases the frequency and up-regulates cell survival genes in CD25+CD4+ TR cells.</a> Int Immunol. 18, 1265-1277 (2006).</li><li>Ascon, D. B., 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=Normal+mouse+kidneys+contain+activated+and+CD3+CD4-+CD8-+double-negative+T+lymphocytes+with+a+distinct+TCR+repertoire.">Normal mouse kidneys contain activated and CD3+CD4- CD8- double-negative T lymphocytes with a distinct TCR repertoire.</a> J Leukoc Biol. 84, 1400-1409 (2008).</li><li>Hamad, A. R., 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=B220++double-negative+T+cells+suppress+polyclonal+T+cell+activation+by+a+Fas-independent+mechanism+that+involves+inhibition+of+IL-2+production.">B220+ double-negative T cells suppress polyclonal T cell activation by a Fas-independent mechanism that involves inhibition of IL-2 production.</a> J Immunol. 171, 2421-2426 (2003).</li><li>Rabb, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiological+role+of+T+lymphocytes+in+renal+ischemia-reperfusion+injury+in+mice.">Pathophysiological role of T lymphocytes in renal ischemia-reperfusion injury in mice.</a> Am J Physiol-Renal. 279, 525-531 (2000).</li><li>Burne, M. J., 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=Identification+of+the+CD4(+)+T+cell+as+a+major+pathogenic+factor+in+ischemic+acute+renal+failure.">Identification of the CD4(+) T cell as a major pathogenic factor in ischemic acute renal failure.</a> J Clin Invest. 108, 1283-1290 (2001).</li><li>Yokota, N., Daniels, F., Crosson, J., Rabb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+T+cell+depletion+in+murine+renal+ischemia-reperfusion+injury.">Protective effect of T cell depletion in murine renal ischemia-reperfusion injury.</a> Transplantation. 74, 759-763 (2002).</li><li>Crispin, J. 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W., 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=FcR+gamma+presence+in+TCR+complex+of+double-negative+T+cells+is+critical+for+their+regulatory+function.">FcR gamma presence in TCR complex of double-negative T cells is critical for their regulatory function.</a> J Immunol. 177, 2250-2257 (2006).</li></ol>

Die Autoren haben nichts zu offenbaren.

Es gibt immer mehr Interesse an DN-T-Zellen, da sie, die in verschiedenen pathologischen Bedingungen, wie Autoimmunerkrankungen, Krebs, Transplantat-Toleranz und primären Erkrankungen der Niere einschließlich akuten Nierenschädigung (AKI) in Verbindung gebracht, Glomerulonephritis 8, 13. Daher besteht Bedarf, die pathophysiologische Funktionen DN-T-Zellen besser zu verstehen und zu charakterisieren. Derzeit jedoch mangelt es in Verständnis der Funktion dieser Zellen im Vergleich zu CD4 und CD8 T-Zellen. E...

A correction was made to: <a href="http://jove.unicartagenaproxy.elogim.com/video/51192/isolation-of-double-negative-t-cells-from-the-kidney">Isolation of Double Negative &#945;&#946; T Cells from the Kidney</a>.
There was an error with an author's name. The author's given name had a typo, this was corrected to:
Samatha Bandapelle
instead of:
Samantha Bandapelle

A correction was made to: Isolation of Double Negative &#945;&#946; T Cells from the Kidney.

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

Periphere αβTCR + CD3 + CD4 - CD8 - doppelt-negative (DN) T-Zellen werden in verschiedene Untergruppen, die unterschiedliche Phänotypen und Funktionen besitzen 1-4 unterteilt. DN-T-Zellen sind wenig verstanden, aber zunehmend auch in pathophysiologischen Immunantworten in verschiedenen Krankheitsmodellen 4-6 verwickelt. DN-T-Zellen sind eine einzigartige Immunzelltyp, der immer an der Regulation von verschiedenen Immun-und Autoimmunreaktionen und die Modulation der Allotransplantat-Toleranz in Verbindung gebracht wird. 4-6, 9 Sie sind selten im peripheren Blut und sekundären lymphatischen Organen (Milz und Lymphknoten ). Allerdings sind sie großen Bewohner der normalen Nieren-und Darmepithel 10-12. Sehr wenig ist über ihre Funktion 7 in der Niere im stationären Zustand und unter pathologischen Bedingungen, wie akute Nierenschädigung (AKI) mit Nieren Transpl. verbundenen bekanntAmeise. Wegen der komplizierten Rollen der verschiedenen Immunzellen bei der Regulierung der Immunantworten einschließlich alloresponses, die Definition der Rolle der einzelnen Spieler ist entscheidend für das Verständnis alloresponses und Gestaltung neuer Therapeutika. Angesichts der erheblichen Zahl von DN-T in der Niere unter physiologischen und unterschiedliche Krankheitszustände vorliegenden Zellen sind DN-T-Zellen wahrscheinlich eine entscheidende Rolle bei der Regulierung der Immun-und Autoimmunreaktionen in Mäusen und Menschen, und alloresponses bei Transplantatempfängern spielen. Thesaurierend wenn auch noch zerstreut Beweise bringen DN-T-Zellen in beiden pathogenen und immunsuppressive Funktionen, aber es ist schlecht verstanden, warum und wie sie eine spezifische schädliche oder drück Funktion aufweisen und wie die Umwelt beeinflusst sie. Aufgrund ihrer geringen Menge in der Niere, sind verbesserte Verfahren zur Isolierung erforderlich. Derzeit gibt es keine Standard-Protokoll für die Isolierung von DN T-Zellen von ter nicht-lymphatischen Geweben. Unser Protokoll beschreibt ein neues Verfahren zur Isolierung von DN-T-Zellen aus der Niere; Jedoch kann das Verfahren auch für verschiedene nicht-lymphoiden Geweben verwendet werden.

<table border="0" cellpadding="0" cellspacing="0" width="1172">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Laminar flow hood&#160;</td>
			<td style="width:257px;">Baker Company, Inc</td>
			<td style="width:276px;"></td>
			<td style="width:200px;">Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Hemocytometer</td>
			<td>Electron Microscopy Sciences</td>
			<td style="width:276px;">63514-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Atmosphere-controlled incubator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td style="width:200px;">&#160;(37&#176;C with 5% CO2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Microscope&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Analytical flow cytometer</td>
			<td style="width:257px;">LSR II&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:439px;">Name of the reagent</td>
			<td style="width:257px;">Company&#160;</td>
			<td style="width:276px;">Catalog&#160; Number</td>
			<td style="width:200px;">Comments&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">RPMI 1640</td>
			<td style="width:257px;">Media Tech</td>
			<td style="width:276px;">10-040-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Collagenase D</td>
			<td style="width:257px;">Roche</td>
			<td style="width:276px;">11088858001</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Percoll&#160;</td>
			<td style="width:257px;">GE Healthcare</td>
			<td style="width:276px;">&#160;17-0891-01</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fetal bovine serum&#160;</td>
			<td>Corning Cellgro</td>
			<td>35-011-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">0.1% sodium azide (NaN3)</td>
			<td style="width:257px;">Sigma-Aldrich&#160;</td>
			<td style="width:276px;">S2002</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Buffer phosphate buffered saline (PBS)</td>
			<td style="width:257px;">Corning Cellgro</td>
			<td>21-040-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">PBS 10X</td>
			<td>Mediatech</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">EDTA buffer&#160;</td>
			<td style="width:257px;">Sigma-Aldrich&#160;</td>
			<td style="width:276px;">E1161</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">LS columns&#160;</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">CD45+ microbeads</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">6.78E-51</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Biotin microbeads&#160;</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">130-090-485</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD45 (clone: 30-F11)&#160; PerCP&#160;</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td style="width:276px;">G1397</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD45 (clone: 30-F11)&#160; APC-Cy7</td>
			<td style="width:257px;">Biolegend&#160;&#160;</td>
			<td style="width:276px;">103116</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-TCR Pacific Blue&#160; (ab-chain, clone: H57-597)</td>
			<td style="width:257px;">Invitrogen</td>
			<td style="width:276px;">HM3628</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD4 PE</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td>12-0043</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD8 FITC</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td>8011-0087</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD4 biotinylated (GK1.5)</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553728</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD8 biotinylated&#160; (clone 53-6.7)</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553029</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Fc receptor (CD16/32) biotinylated</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553143</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-MHC class II (I-A d) biotinylated</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553609</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD1d PBS-57 tetramer</td>
			<td style="width:257px;">NHI tetramer&#160;</td>
			<td style="width:276px;">lote # 15621-PBS57</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:439px;">AutoMACS Running Buffer-&#160; MACS Separation Buffer&#160;</td>
			<td style="width:257px;">MIlteny I Biotec</td>
			<td style="width:276px;">130-091-221</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">C57BL/6J mice</td>
			<td>Jackson Labs</td>
			<td style="width:276px;">000664</td>
			<td>Kidneys - Lymph Nodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri dish&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:276px;">356517</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">70mc filter&#160;</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plunger&#160;</td>
			<td style="width:257px;"></td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GeneMate Tubes 50 ml</td>
			<td>Bioexpress</td>
			<td>C-3394-3</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">GeneMate Tubes 15 ml</td>
			<td>Bioexpress</td>
			<td style="width:276px;">C-3394-1</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri Dish&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:276px;">S33580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plate 24 wells&#160;</td>
			<td style="width:257px;">BD Biosciences</td>
			<td style="width:276px;">354723</td>
			<td style="width:200px;"></td>
		</tr>
	</tbody>
</table>

isolation of double negative &#945;&#946; t cells from the kidney

Derzeit gibt es keine Standard-Protokoll für die Isolierung von DN-T-Zellen von den nicht-lymphatischen Gewebe trotz ihrer zunehmend berichtet Beteiligung an verschiedenen Immunantworten. DN-T-Zellen sind eine einzigartige Immunzelltyp, der bei der Regulierung der Immun-und Autoimmunreaktionen und Toleranz zu allotransplants 1-6 gebracht wurde. DN-T-Zellen sind jedoch selten im peripheren Blut und sekundären lymphatischen Organen (Milz und Lymphknoten), jedoch sind die Haupt Bewohner der normalen Niere. Sehr wenig ist über die pathophysiologische Funktion 7 aufgrund ihrer Mangel in dem Umfang bekannt. Wir haben vor kurzem beschrieben, eine umfassende phänotypische und funktionelle Analyse dieser Bevölkerung in der Niere 8 in stationären und während der Ischämie Reperfusion. Analyse der DN-T-Zell-Funktion wird stark durch die Entwicklung eines Protokolls für ihre Isolation von der Niere verbessert werden. Hier ein neues Protokoll, das ermöglicht isolatio beschreiben wirn von hochreinem ab CD4 + CD8 + T-Zellen und DN-T-Zellen aus der Maus-Nieren. Kurz gesagt, wir verdauen Nierengewebe mittels Collagenase und zu isolieren, Nierenmononuklearen Zellen (KMNC) durch Dichtegradientenzentrifugation. Dies wird durch zwei Schritte auf hämatopoetischen T-Zellen von 3% bis 70%, gefolgt von KMNC bereichern. Der erste Schritt besteht aus einer positiven Selektion von hämatopoetischen Zellen mit einer CD45 +-Isolierungs-Kit. Im zweiten Schritt werden negativ DN-T-Zellen durch Entfernen von nicht-gewünschten Zellen mit CD4, CD8, MHC Klasse II und monoklonale Antikörper und CD1d α-GalCer Tetramer isoliert. Diese Strategie führt zu einer Bevölkerung von mehr als 90% rein DN T-Zellen. Oberflächenfärbung mit den oben erwähnten Antikörper, gefolgt von FACS-Analyse wird verwendet, um die Reinheit zu bestätigen.

Wildtyp C57BL / 6 (B6) Niere enthält etwa 1,5 bis 2,1 x 10 6 mononuklearen Zellen pro Niere. Weniger als etwa 10% hämatopoetische CD45 +-Zellen. Für die Herstellung von Nieren mononukleären Zellen (KMNC) werden die Nieren in kleine Stücke geschnitten, wie in Fig. 1 gezeigt, gefolgt von Verdauung mit Collagenase 5%. Dies wird durch Durchführen einer Dichte-Gradienten-Zentrifugation auf KMNC Schicht (Fig. 2, links) zu sammeln, gefol...

Watch this Scientific Journal Video about Isolation of Double Negative ÃŽÂ±ÃŽÂ² T Cells from the Kidney at JoVE.com

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

DNabT Zellen sind selten unter peripheren T-Zellen; aber reichlich sind sie in bestimmten Nicht-lymphatischen Gewebe. Schwierigkeitsgrad der Isolierung DN T-Zellen von nicht-lymphatischen Gewebe behindert ihre Funktionsanalyse trotz steigender erkannt pathophysiologische Bedeutung. Wir beschreiben eine neue Methode zur Isolierung von hoch gereinigten DN T-Zellen aus murinen Niere.

Isolierung von Double Negative αβ T-Zellen aus der Niere

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-double-negative-t-cells-from-the-kidney

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

12.1K Aufrufe.  Johns Hopkins University School of Medicine.  DNabT Zellen sind selten unter peripheren T-Zellen; aber reichlich sind sie in bestimmten Nicht-lymphatischen Gewebe. Schwierigkeitsgrad der Isolierung DN T-Zellen von nicht-lymphatischen Gewebe behindert ihre Funktionsanalyse trotz steigender erkannt pathophysiologische Bedeutung. Wir beschreiben eine neue Methode zur Isolierung von hoch gereinigten DN T-Zellen aus murinen Niere. 

Serie: Isolation of Double Negative αβ T Cells from the Kidney

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generation der induzierte regulatorische T-Zellen aus primären humanen naiven und Gedächtnis-T-Zellen

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Forschung

Immunologie und Infektion

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.

Isolierung und Charakterisierung von dendritischen Zellen und Makrophagen aus dem Mausdarm

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

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Isolierung von Precursor B-Zell-Subsets aus Nabelschnurblut

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Isolierung und Differenzierung von Th17 Naive CD4-T-Lymphozyten

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Ableitung von T-Zellen<em&gt; In-vitro-</em&gt; Aus embryonalen Stammzellen der Maus

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Generation of Human Alloantigen-spezifischen T-Zellen aus peripherem Blut

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Maus Naive CD4<sup&gt; +</sup&gt; T-Zell-Isolierung und<em&gt; In-vitro-</em&gt; Differenzierung in T-Zell-Populationen

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T-Zellen erfassen Bakterien durch Transinfection von dendritischen Zellen

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Effiziente Isolation Protokoll für die B- und T-Lymphozyten von Menschengaumenmandeln

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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.

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.

Isolierung von Sertoli-Zellen und peritubulären Zellen aus Ratte Hoden

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 ...

Isolierung von Double Negative αβ T-Zellen aus der Niere

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