Name: an in vivo mouse model to measure na&#239;ve cd4 t cell activation, proliferation and th1 differentiation induced by bone marrow-derived dendritic cells
Uploaded: 2018-08-22T15:00:00
Description: Watch this Scientific Journal Video about An In Vivo Mouse Model to Measure NaÃ¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells at JoVE

Dr. Simon Bartlett İngilizce düzenleme için teşekkür ediyoruz. Bu çalışmada: Instituto de hibe tarafından desteklenmiştir Salud Carlos III (ISCIII) (PI14/00526; PI17/01395; CP11/00145; CPII16/00022), Miguel Servet Program ve Fundación Ramón Areces; Fondo Europeo de Desarrollo bölgesel (FEDER) ortak fon ile. CNIC Ekonomi Bakanlığı, sanayi ve rekabet (MEIC) ve Pro CNIC Foundation tarafından desteklenen ve bir Severo Ochoa Center of Excellence (SEV-2015-0505) olduğunu. RTF Fundación Ramón Areces ve CNIC, VZG ISCIII, Instituto de birimi BHF tarafından kurulan Investigación Sanitaria hastane 12 de Octubre (imas12) ve JMG-G ISCIII Miguel Servet Program ve imas12.

Deneysel prosedürler Fundación Centro Nacional de tarafından onaylanmış Investigaciones Cardiovasculares Carlos III (CNIC) ve Comunidad Autonoma de Madrid İspanya ve Avrupa yönergelere uygun olarak. Fare belirli patojen ücretsiz (SPF) koşullarında yetiştirilen ve karbondioksit (CO2) inhalasyon tarafından euthanized.
1. yalıtım yırtılmalarıteşhis ve uyluk fare kemik iliği hücrelerinin
Not: Vahşi tipli C57BL suşlarının CD45.2 veya Ly5....

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J., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector+and+memory+T-cell+differentiation:+implications+for+vaccine+development.">Effector and memory T-cell differentiation: implications for vaccine development.</a> Nature Reviews Immunology. 2 (4), 251-262 (2002).</li><li>Iborra, S., Gonzalez-Granado, J. 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Bu iletişim kuralı etkinleştirme, yayılması ve saf CD4 T hücrelerinin farklılaşma modüle için BM kaynaklı DCs kapasitesini karakterizasyonu için izin verir. Ayrıca, aynı zamanda CD4 T hücrelerinin modülasyonu karşı hassasiyeti değerlendirmek için BM kaynaklı DC'ler tarafından kullanılabilmesi için. Bu iletişim kuralı ile ölçülen vivo içindebu olaylar değişimler olabilir.
Soruşturma altında hipotez bağlı olarak, T hücreleri ve DCs çeşitli bileşim...

CD4 T hücreleri ve antijen sunan hücreler (ZPT) DCs gibi bağışıklık mikrobiyal patojenler1,2için gerekli ortam araçları vardır. Periferik lenfoid organlarda CD4 T hücreleri belirli antijenleri ZPT3,4,5tarafından sunulan tanınması üzerine aktif hale gelir. Aktif CD4 T hücreleri çoğalırlar ve doğru edinilmiş bağışıklık yanıtı6,7gelişimi için gerekli olan farklı belirli efektör Th hücrelerine ayırt etmek. Bu işlemlerin kontrolünü zararlı doku hasarı8üretmeden patojen öldürür yeterli bir adaptif savunma üretmek için önemlidir. İnci hücreleri ifade veya yüzey molekülleri, transkripsiyon faktörleri ve efektör sitokinlerin üretimini göre tanımlanır ve önemli ve hassas fonksiyonları yanıt patojenlerin1. Hücre Th1 hücre alt transkripsiyon faktörü T-bahis ve sitokin interferon γ (IFNγ) hızlı ve intrasellüler patojenlerin1karşı ana savunma katılmak. Miktar saf CD4 T Hücre aktivasyonu, yayılması ve Th1 farklılaşma rol T hücreleri oyunda bir bağışıklık yanıtı değerlendirirken yararlı bir araçtır.
Bu iletişim kuralı sağlar içinde vivo çözümleme kapasiteleri için in vitro -BM kaynaklı DCs harekete geçirmek, yayılması ve saf CD4 T hücrelerinin Th1 farklılaşma modüle oluşturulan. İletişim kuralı da saf CD4 T hücreleri aktive, çoğalırlar indüklenen için kapasitesini değerlendirmek için hizmet vermektedir ve Th1 (Şekil 1) farklı. Bu çok yönlü iletişim kuralı yetersizlik özellikle işlemek veya tamamen vivo içinde iletişim kuralları'nda okudu hücre nüfus seçin kaçınmanızı sağlar. Çeşitli moleküller ve DCs tedavisi etkileri genetiği değiştirilmiş fare5 BM kullanarak veya tedavi ya da genetik olarak izole BM hücreleri9manipüle okudu olabilir. Benzer şekilde, T hücre yanıt-e doğru farklı kaynaklardan veya çeşitli manipülasyonlar3,8,10sonra T hücreleri evlat edinen transfer için alma tarafından keşfedilmeyi olabilir.
İki kat bu protokolü başlıca avantajları şunlardır. T Hücre aktivasyonu, yayılmasını önleme ve Th1 farklılaşma bir akış sitometresi yaklaşımla analiz edilir; ve bu böylece vitro oluşabilecek değişiklikler önlenmesi ve hücre tipleri ve sadece sağlam organları11' de bulunan diğer faktörler de dahil olmak üzere in vivo çalışmalar ile birlikte yürür.
Hayati boya kullanımı radyoaktivite kullanımı kaçınırken hücre çoğalması izlemek için yaygın olarak kullanılan bir tekniktir. Nükleer silahların yayılmasına karşı bu kimyasalları ile ölçüm boya seyreltme hücre bölünmesi sonra temel alır. Ayrıca, bu boyalar birden çok dalga boylarında tespit edilebilir ve kolayca birden çok floresan antikor veya işaretleri ile akış sitometresi tarafından incelenir. Biz bu protokolü yardımcı programını nasıl T Hücre aktivasyonu, yayılmasını önleme ve Th1 farklılaşma akış sitometresi tarafından çözümlenebilir göstererek vurgulayın.

<table>
	<tbody>
		<tr>
			<td>Ethanol</td>
			<td>VWR Chemicals</td>
			<td>20,824,365</td>
			<td>5 L</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>11000-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>11253-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>10020-00</td>
			<td>Box of 100 blades</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>SIGMA</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>LONZA</td>
			<td>DE17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute medium (RPMI)</td>
			<td>GIBCO</td>
			<td>21875-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>25-gauge needle</td>
			<td>BD Microlance 3</td>
			<td>300600</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Novico</td>
			<td>N15663</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m nylon web filter</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood lysis buffer</td>
			<td>SIGMA</td>
			<td>R7757</td>
			<td>100 Ml</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>SIGMA</td>
			<td>ED2SS-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>A7906</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>SIGMA</td>
			<td>302643-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture-plates</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Cambrex</td>
			<td>BE17-737E</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Merck</td>
			<td>8-05740-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>LONZA</td>
			<td>BE13-115E</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>LONZA</td>
			<td>BE17-605E</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine Granulocyte Macrophage colony-stimulating factor (GM-CSF)</td>
			<td>Prepotech</td>
			<td>315-03</td>
			<td></td>
		</tr>
		<tr>
			<td>lipopolysaccharide (LPS)</td>
			<td>SIGMA-ALDRICH</td>
			<td>L2018</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well-plate</td>
			<td>Costar</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>v450 anti-mouse CD11b antibody</td>
			<td>BD Biosciences</td>
			<td>560455</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD64 antibody</td>
			<td>BioLegend</td>
			<td>139303</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD115 antibody</td>
			<td>BioLegend</td>
			<td>135505</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD11c antibody</td>
			<td>BioLegend</td>
			<td>117305</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse MHCII antibody</td>
			<td>BioLegend</td>
			<td>125507</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD86 antibody</td>
			<td>BioLegend</td>
			<td>105011</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD80 antibody</td>
			<td>BioLegend</td>
			<td>104713</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry tubes</td>
			<td>Zelian</td>
			<td>300800-1</td>
			<td>PS 12X75 5mL</td>
		</tr>
		<tr>
			<td>OTII Ovoalbumine peptide</td>
			<td>InvivoGen</td>
			<td>323-339</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD8&#945; antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0081-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated IgM antibody</td>
			<td>BioLegend</td>
			<td>406503</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated B220 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0452-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD19 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0193-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated MHCII (I-Ab) antibody</td>
			<td>BioLegend</td>
			<td>115302</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD11b antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0112-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD11c antibody</td>
			<td>BioLegend</td>
			<td>117303</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD44 antibody</td>
			<td>BioLegend</td>
			<td>103003</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD25 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0251-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated DX5 antibody</td>
			<td>BioLegend</td>
			<td>108903</td>
			<td></td>
		</tr>
		<tr>
			<td>streptavidin-coated magnetic microbeads</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic cell separator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td>QuadroMACS Separator</td>
		</tr>
		<tr>
			<td>Separation columns</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD4 antibody</td>
			<td>BioLegend</td>
			<td>100408</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD3 antibody</td>
			<td>BioLegend</td>
			<td>100235</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD8 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>35-0081-U025</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Violet Tracer</td>
			<td>Thermofisher</td>
			<td>C34557</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD25 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-0251-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa647 anti-mouse CD69 antibody</td>
			<td>BioLegend</td>
			<td>104518</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCPCY5.5 anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>65-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>PECY7 anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>60-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD45.2 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>35-0454</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>SIGMA-ALDRICH</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12 Myristate 13 Acetate (PMA)</td>
			<td>SIGMA</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A (BD GolgiPlug)</td>
			<td>BD</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Millipore</td>
			<td>8-18715-02100</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracellular permeabilization buffer</td>
			<td>eBioscience</td>
			<td>00-8333</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse IFNg antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-7311-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-block (anti-mouse CD16/CD32)</td>
			<td>Tonbo Biosciences</td>
			<td>70-0161-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.SJL CD45.1 mice</td>
			<td>The Jackson Laboratory</td>
			<td>002014</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRFortessa&#8482; Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td>649225</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI Solution</td>
			<td>Thermofisher</td>
			<td>62248</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo mouse model to measure na&#239;ve cd4 t cell activation, proliferation and th1 differentiation induced by bone marrow-derived dendritic cells

Miktar (Th1) saf CD4 T Hücre aktivasyonu, yayılması ve farklılaşma T yardımcı 1 için hücreleri T hücreleri bir bağışıklık yanıtındaki oynadığı rol değerlendirmek için yararlı bir yoldur. Bu iletişim kuralı granülosit makrofaj koloni uyarıcı faktör (GM-CSF) türetilmiş-dendritik hücreler (DC) elde etmek için kemik iliği (BM) ataları vitro farklılaşma açıklar. Protokol ayrıca ovalbumin peptid (OVAp) evlat edinen transferini açıklanır-GM-CSF-türetilmiş DCs ve saf CD4 T hücreleri OTII transgenik fareler vivo içinde harekete geçirmek, yayılması ve Th1 farklılaşma çözümlemek amacıyla yüklü Transfer edilen CD4 T hücreleri. Bu iletişim kuralı tamamen in vivo Yöntemler özellikle işlemek veya okudu hücre nüfus seçmek için yetersizlik tarafından dayatılan sınırlandırılması kaçınmanızı sağlar. Ayrıca, bu protokol çalışmaları böylece değişiklikler vitro oluşabilir fonksiyonel etkenler ve hücre tipleri ve sadece olduğu gibi organlarda bulunan diğer faktörlerin etkisi kaçınarak bir in vivo ortamda olanak sağlar. Protokol değişiklikleri DCs içinde oluşturmak için yararlı bir araçtır ve T hücreleri edinilmiş bağışıklık yanıtı, potansiyel olarak kökenli veya çok sayıda bağışıklık ilişkili hastalıkları gelişimi anlamak için önemli sonuçları sağlamanın değiştirin.

Şekil 1 bu protokol için açıklanan adımları gösterilmektedir. Şekil 2 yalıtım ve fare BM hücre kültür gösterilmektedir. GM-CSF ve LPS eklenmesi için bu kültürler vitro üretimi sağlar ve farklılaşma Akış Sitometresi Analizi ve elde edilen DCs. OVAp yüklü GM-CSF BM kaynaklı DCs olgunlaşma DCs. Şekil 3 olgunlaşma gösterilmektedir ve izole hayati hücre izleme boya ...

Watch this Scientific Journal Video about An In Vivo Mouse Model to Measure NaÃ¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells at JoVE

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Burada, bir protokol saf CD4 T hücre (T hücre) etkinleştirme, yayılması ve GM-CSF kemik iliği (BM) tarafından indüklenen Th1 farklılaşma vivo içinde belirlenmesi için mevcut-dendritik hücreler (DC) türetilmiş. Ayrıca, bu iletişim kuralını BM ve T-hücre izolasyon, DC üretimi ve DC ve T hücreli evlat edinen transfer açıklar.

Vivo fare modeli ölçü saf CD4 T Hücre aktivasyonu, yayılması ve kemik iliği türevi dendritik hücreler tarafından indüklenen Th1 farklılaşma

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

This method can help answer key questions in the immunology field about T-cell activation, proliferation, and Th1 differentiation, as well antigen presenting cell stimulation of adaptive immunity. The main advantage of this technique is that it allows the study of an in vivo environment while simultaneously allowing cell manipulation in vitro. Visual demonstration of this method is critical as the organ and bone isolation and adoptive transfer steps are difficult to master through text instructions alone.For bone marrow cell isolation, disinfect the hind limbs of an adult mouse with 70%ethanol and use scissors to make a lateral skin incision across the hind limbs. Insert scissors at the medial aspect of the legs, and cut the skin with the scissors until the beginning of the hind limbs. Then use forceps to separate the skin from the legs.Using scissors, separate the muscle from the femur and tibia, and use scissors to disarticulate the hip joint, breaking the femur head. Then separate the femure from the tibia without breaking the bone ends, and place the bones in a petri dish of complete medium on ice. When all of the bones have been harvested, carefully cut off the proximal and distal ends of each bone with a scalpel, and use a 1 millileter syringe equipped with a 25-guage needle to flush the bones repeatedly with a total volume of 10 millileters of warm complete RPMI medium.When all of the bones have been flushed, transfer the entire contents of the dish to a 50-millileter conical tube fitted with a 70-micrometer pore nylon web filter, and carefully dislodge any debris and cell conglomerates with gentle stirring and pipetting. Collect the bone marrow cells by centrifugation. Resuspend the pellet in one millileter of four-degree Celsius red blood cell lysis buffer with pipetting, and incubate the mix on ice for five minutes.At the end of the incubation, stop the lysis with 10 millileter of PVS, and resuspend the pellet in 25 millileters of complete medium for a second centrifugation. Then resuspend the cells in 5 millileters of fresh complete medium for counting, and centrifuge the cells again. For secondary lymphoid tissue cell isolation, collect the inguinal, axillary, brachial, cervical, and mesentaric lymph nodes, and spleen from OT-II transgenic mice, and transfer the secondary lymphoid tissues into a plastic petri dish containing 10 millileters of complete RPMI medium.When all of the tissues have been harvested, transfer the lymph nodes and spleen to a 70-micrometer cell strainer in a 50-millileter conical tube, and use a syringe plunger to homogenize the tissues. Flush the strainer with up to 15 millileters of medium, and spin down the cells by centrifugation. Then isolate the CD4-positive T cells by magnetic bead sorting, according to standard protocols.For in vivo activation of the naive OT-II CD4-positive T-cells, label the magnetic bead-isolated T-cells with vital cell tracer dye, according to standard protocols, and resuspend the labeled cells at a 10 to the sixth cells per 100 microliters of PBS concentration. Then adoptively transfer 100 microliters of cells per animal via the tail vein. Pre-warm the mice before the injection in order to ensure that the tail veins are enough dilated to allow delivery of entire cell volume.After 24 hours, deliver 10 to the fifth LPS-matured OVA peptide-loaded dendritic cells by subcutaneous injection into the foot pad of each T-cell injected animal. Two and five days after the adoptive challenge, harvest the poplitial lymph nodes from the recipient animals for tissue processing and T-cell isolation, as demonstrated. To analyze T-cell activation, stain day two-isolated cells with fluorescent antibodies against CD4, CD69, and CD25 to determine the percentage of CD64-positive, CD25-positive T-cells within the recipient CD4-positive population by flow cytometry.To assess T-cell proliferation, stain day five-isolated cells with the appropriate fluorescent antibodies against CD4, and analyze the decay of the vital cell tracer dye signal in the donor CD4-positive T-cell population by flow cytometry. To evaluate Th1 differentiation, plate four times 10 to the fifth day five-isolated T-cells per well in 200 microliters of complete medium, supplemented with ionomycin and PMA per well, in a 96-well plate. Then place the plate in a 37 degree Celsius cell culture incubator for six hours, adding Brefeldin A to each well to block cytokine secretion during the last four hours of the stimulation.At the end of the incubation, centrifuge the plate, and discard the supernatant by rapid inversion. Stain the cells with anti-CD4 antibody, followed by fixation in 100 microliters of 2%paraformaldehyde and 1%sucrose for 20 minutes at room temperature. Permeabilize the cells with 50 microliters of permeabilization buffer for 30 minutes at 4 degrees Celsius, followed by a centrifugation wash with 150 microliters of PBS per well.Next, stain for the intracellular cytokines of interest, according to standard protocols for 30 minutes at room temperature, followed by two centrifugation washes with 150 microliters of PBS supplemented with 1%saponin per well. Then, analyze the cells by flow cytometry. Flow cytometric analysis of GM-CSF bone marrow-derived dendritic cells after LPS stimulation, reveals an upregulation of maturation markers, such as MHCII, CD80, and CD86.Naive CD4 positive OT-II T-cells become activated after adoptive transfer to recipient animals by their upregulation of T-cell activation markers surface expression, and multiple rounds of cell division. Upon activation, the proliferating CD-positive OT-II T-cells demonstrate a Th1 phenotype, as demonstrated by their intracellular interferon gamma expression. Once mastered, this technique can be completed in eight hours if it's performed properly.While attempting this procedure, it's important to remember to prepare all the reagents and materials the day before the experiment. After its development, this technique paved the way for researchers in immunology to study dendritic cells and T-cells in mice. After watching this video, you should have a good understanding of how to isolate bone marrow from the femurs and tibia of mice, and how to transfer T-cells and dendritic cells to recipient animals.Don't forget that working with sharp instruments such needles, scalpels, or scissors, can be extremely hazardous, so precautions such as using the appropriate finger protection should always be taken while performing these procedures.

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Fare T-hücrelerinin, T-hücre reseptörleri retroviral İletimi

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

İmmünoloji ve Enfeksiyon

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

İzleme Araştırmaları Qdot nanokristaller murin Kemik İliği-türevli dendritik hücrelerin üretilmesi ve Etiketleme

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Naif CD4 adenoviral İletimi Treg Farklılaşma Eğitim Hücreler T

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

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

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

İzolasyon ve naif CD4 T Lenfositler Th17 Farklılaşma

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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 Enfekte CD4 Doğuştan Algılama Değerlendirilmesi<sup&gt; +</sup&gt; Bir kullanılması plazmositoid dendritik hücreler tarafından T Hücreleri<em&gt; Ex vivo</em&gt; Co-kültür sistemi.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

İzolasyon ve Kemirgen Kemik İliği intravenöz enjeksiyon Türetilmiş Monositler

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

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

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

Fare Naif CD4<sup&gt; +</sup&gt; T Hücre İzolasyonu ve<em&gt; In vitro</em&gt; T hücre alt içine Farklılaşma

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

T hücreleri dendritik hücreler gelen Transinfection tarafından Bakteriler Yakalama

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

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

İzolasyon ve<em&gt; Ex Vivo</emVδ1 of&gt; Kültür<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; γδ T hücrelerinin bir Extrathymic αβT hücre Progenitör

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Bir TGF-β-içeren Protokolü kullanılarak naif CD4 + T hücrelerinden insan CD4 + FoxP3 + Bağlı regülatör T hücreleri (iTregs) in vitro ayrımında