<meta charset="utf8"></head><body>Akış Sitometrisi Kullanılarak Naif CD4+ T Hücrelerinden Th17 Hücre Farklılaşması

<ol>
	<li>Korn, T., Bettelli, E., Oukka, M., Kuchroo, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17+and+Th17+cells.">IL-17 and Th17 cells.</a> Annu Rev Immunol. 27, 485-517 (2009).</li><li>Bhaumik, S., Basu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+dynamics+of+Th17+differentiation+and+its+developmental+plasticity+in+the+intestinal+immune+response.">Cellular and molecular dynamics of Th17 differentiation and its developmental plasticity in the intestinal immune response.</a> Front Immunol. 8, 254 (2017).</li><li>Bettelli, E., Korn, T., Oukka, M., Kuchroo, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+effector+functions+of+T(H)17+cells.">Induction and effector functions of T(H)17 cells.</a> Nature. 453 (7198), 1051-1057 (2008).</li><li>Korn, T., 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=IL-21+initiates+an+alternative+pathway+to+induce+proinflammatory+T(H)17+cells.">IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells.</a> Nature. 448 (7152), 484-487 (2007).</li><li>Bettelli, E., 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=Reciprocal+developmental+pathways+for+the+generation+of+pathogenic+effector+TH17+and+regulatory+T+cells.">Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells.</a> Nature. 441 (7090), 235-238 (2006).</li><li>Yang, X. O., 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=T+helper+17+lineage+differentiation+is+programmed+by+orphan+nuclear+receptors+ROR+alpha+and+ROR+gamma.">T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma.</a> Immunity. 28 (1), 29-39 (2008).</li><li>Ghoreschi, 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=Generation+of+pathogenic+T(H)17+cells+in+the+absence+of+TGF-&#946;+signalling.">Generation of pathogenic T(H)17 cells in the absence of TGF-&#946; signalling.</a> Nature. 467 (7318), 967-971 (2010).</li><li>Lee, Y., 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=Induction+and+molecular+signature+of+pathogenic+TH17+cells.">Induction and molecular signature of pathogenic TH17 cells.</a> Nat Immunol. 13 (10), 991-999 (2012).</li><li>McGeachy, 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=The+interleukin+23+receptor+is+essential+for+the+terminal+differentiation+of+interleukin+17-producing+effector+T+helper+cells+in+vivo.">The interleukin 23 receptor is essential for the terminal differentiation of interleukin 17-producing effector T helper cells in vivo.</a> Nat Immunol. 10 (3), 314-324 (2009).</li><li>Wu, 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=The+TGF-&#946;+superfamily+cytokine+Activin-A+is+induced+during+autoimmune+neuroinflammation+and+drives+pathogenic+Th17+cell+differentiation.">The TGF-&#946; superfamily cytokine Activin-A is induced during autoimmune neuroinflammation and drives pathogenic Th17 cell differentiation.</a> Immunity. 54 (2), 308-323 (2021).</li><li>Ramesh, 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=Pro-inflammatory+human+Th17+cells+selectively+express+P-glycoprotein+and+are+refractory+to+glucocorticoids.">Pro-inflammatory human Th17 cells selectively express P-glycoprotein and are refractory to glucocorticoids.</a> J Exp Med. 211 (1), 89-104 (2014).</li><li>Basdeo, S. 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=Ex-Th17+(nonclassical+Th1)+cells+are+functionally+distinct+from+classical+Th1+and+Th17+cells+and+are+not+constrained+by+regulatory+T+cells.">Ex-Th17 (nonclassical Th1) cells are functionally distinct from classical Th1 and Th17 cells and are not constrained by regulatory T cells.</a> J Immunol. 198 (6), 2249-2259 (2017).</li><li>Cua, D. 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=Interleukin-23+rather+than+interleukin-12+is+the+critical+cytokine+for+autoimmune+inflammation+of+the+brain.">Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain.</a> Nature. 421 (6924), 744-748 (2003).</li><li>Awasthi, 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=Cutting+edge:+IL-23+receptor+gfp+reporter+mice+reveal+distinct+populations+of+IL-17-producing+cells.">Cutting edge: IL-23 receptor gfp reporter mice reveal distinct populations of IL-17-producing cells.</a> J Immunol. 182 (10), 5904-5908 (2009).</li><li>J&#228;ger, A., Dardalhon, V., Sobel, R. A., Bettelli, E., Kuchroo, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th1,+Th17,+and+Th9+effector+cells+induce+experimental+autoimmune+encephalomyelitis+with+different+pathological+phenotypes.">Th1, Th17, and Th9 effector cells induce experimental autoimmune encephalomyelitis with different pathological phenotypes.</a> J Immunol. 183 (11), 7169-7177 (2009).</li><li>Lee, Y., Collins, M., Kuchroo, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+targets+and+triggers+of+autoimmunity.">Unexpected targets and triggers of autoimmunity.</a> J Clin Immunol. 34, S56-S60 (2014).</li><li>Chang, D., 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=The+conserved+non-coding+aequences+CNS6+and+CNS9+control+cytokine-induced+Rorc+transcription+during+T+helper+17+cell+differentiation.">The conserved non-coding aequences CNS6 and CNS9 control cytokine-induced Rorc transcription during T helper 17 cell differentiation.</a> Immunity. 53 (3), 614-626 (2020).</li><li>Bunte, K., Beikler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th17+cells+and+the+IL-23/IL-17+axis+in+the+pathogenesis+of+periodontitis+and+immune-mediated+inflammatory+diseases.">Th17 cells and the IL-23/IL-17 axis in the pathogenesis of periodontitis and immune-mediated inflammatory diseases.</a> Int J Mol Sci. 20 (14), 3394 (2019).</li><li>Zhao, Y., Liu, Z., Qin, L., Wang, T., Bai, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+mechanisms+of+Th17+differentiation+and+the+Yin-Yang+of+Th17+cells+in+human+diseases.">Insights into the mechanisms of Th17 differentiation and the Yin-Yang of Th17 cells in human diseases.</a> Mol Immunol. 134, 109-117 (2021).</li><li>Berghmans, N., 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=Interferon-&#947;+orchestrates+the+number+and+function+of+Th17+cells+in+experimental+autoimmune+encephalomyelitis.">Interferon-&#947; orchestrates the number and function of Th17 cells in experimental autoimmune encephalomyelitis.</a> J Interferon Cytokine Res. 31 (7), 575-587 (2011).</li><li>Wu, B., Wan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+control+of+pathogenic+Th17+cells+in+autoimmune+diseases.">Molecular control of pathogenic Th17 cells in autoimmune diseases.</a> Int Immunopharmacol. 80, 106187 (2020).</li><li>Du, 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=Growth+hormone+releasing+hormone+signaling+promotes+Th17+cell+differentiation+and+autoimmune+inflammation.">Growth hormone releasing hormone signaling promotes Th17 cell differentiation and autoimmune inflammation.</a> Nat Commun. 14 (1), 3298 (2023).</li><li>Ernst, D. N., Weigle, W. O., Noonan, D. J., McQuitty, D. N., Hobbs, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+age-associated+increase+in+IFN-gamma+synthesis+by+mouse+CD8++T+cells+correlates+with+shifts+in+the+frequencies+of+cell+subsets+defined+by+membrane+CD44,+CD45RB,+3G11,+and+MEL-14+expression.">The age-associated increase in IFN-gamma synthesis by mouse CD8+ T cells correlates with shifts in the frequencies of cell subsets defined by membrane CD44, CD45RB, 3G11, and MEL-14 expression.</a> J Immunol. 151 (2), 575-587 (1993).</li><li>Radtke, A. 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=IBEX:+an+iterative+immunolabeling+and+chemical+bleaching+method+for+high-content+imaging+of+diverse+tissues.">IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues.</a> Nat Protoc. 17 (2), 378-401 (2022).</li><li>El-Hindi, 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=T-cell-specific+CerS4+depletion+prolonged+inflammation+and+enhanced+tumor+burden+in+the+AOM/DSS-induced+CAC+model.">T-cell-specific CerS4 depletion prolonged inflammation and enhanced tumor burden in the AOM/DSS-induced CAC model.</a> Int J Mol Sci. 23 (3), 1866 (2022).</li><li>Cooney, L. A., Towery, K., Endres, J., Fox, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+and+resistance+to+regulation+by+IL-4+during+Th17+maturation.">Sensitivity and resistance to regulation by IL-4 during Th17 maturation.</a> J Immunol. 187 (9), 4440-4450 (2011).</li><li>Zhu, X., 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=A+novel+interleukin-2-based+fusion+molecule,+HCW9302,+differentially+promotes+regulatory+T+cell+expansion+to+treat+atherosclerosis+in+mice.">A novel interleukin-2-based fusion molecule, HCW9302, differentially promotes regulatory T cell expansion to treat atherosclerosis in mice.</a> Front Immunol. 14, 1114802 (2023).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD62L, BV650, clone MEL-14, 1:200 dilution</td>
			<td>BD</td>
			<td>Cat# 564108; RRID: AB_2738597</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat monoclonal anti-CD4, FITC, clone RM4-5, 1:200 dilution</td>
			<td>BioLegend</td>
			<td>Cat#100509; RRID: AB_312712</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat monoclonal anti-IL-17A, PE, clone TC11-18H10.1, 1:200 dilution</td>
			<td>BioLegend</td>
			<td>Cat#506903; RRID: AB_ 315463</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat monoclonal anti mouse/human CD44, APC, clone IM&#38;, 1:200 dilution</td>
			<td>BioLegend</td>
			<td>Cat#103012; RRID: AB_312963</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat monoclonal anti-ROR&#947;T, APC, clone B2D, 1:200 dilution</td>
			<td>Invitrogen</td>
			<td>Cat#17-6981-80; RRID: AB_2573253</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat monoclonal FOXP3 antibody, PE, clone FJK-16s, 1:200 dilution</td>
			<td>Invitrogen</td>
			<td>Cat#12-5773-82; RRID: AB_465936</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals, peptides, and recombinant proteins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD3 SAFIRE purified</td>
			<td>biogems</td>
			<td>Cat#05112-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse CD28 SAFIRE purified</td>
			<td>biogems</td>
			<td>Cat#10312-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IFN gamma</td>
			<td>biogems</td>
			<td>Cat#80822-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse IL-4</td>
			<td>biogems</td>
			<td>Cat#81112-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Xilong scientific</td>
			<td>Cat#64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>Cat#10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>FcR Blocking reagent</td>
			<td>Miltenyi Biotec</td>
			<td>Cat#130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>gibco</td>
			<td>Cat#35050079</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse rIL-1 beta</td>
			<td>Sino Biological</td>
			<td>Cat#50101-MNAE</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse rIL-6</td>
			<td>Sino Biological</td>
			<td>Cat#50136-MNAE</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse rIL-23</td>
			<td>Sino Biological</td>
			<td>Cat#CT028-M08H</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse TGF beta 1</td>
			<td>Sino Biological</td>
			<td>Cat#50698-M08H</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Procell</td>
			<td>Cat#PB180327</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine IL-2</td>
			<td>peprotech</td>
			<td>Cat#212-12</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 with L-glutamine</td>
			<td>Gibco</td>
			<td>Cat#11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin solution</td>
			<td>Gibco</td>
			<td>Cat#15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>Cat#M6250-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Critical commercial assays</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Organ Lymphocyte Separation Solution Kit</td>
			<td>Tbdscience</td>
			<td>Cat#TBD0018SOP</td>
			<td>Contains animal spleen tissue lymphocyte separation liquid, tissue sample diluent (cat no. 2010C1119), sample cleaning solution (cat no. 2010X1118), sample washing solution (cat no. TBTDM-W), tissue homogenate flushing liquid (F2013TBD)</td>
		</tr>
		<tr>
			<td>ChamQ SYBR qPCR Master Mix (High ROX Premixed)</td>
			<td>Vazyme</td>
			<td>Cat#Q341-02</td>
			<td>https://www.vazymebiotech.com/product-center/chamq-sybr-qpcr-master-mix-high-rox-premixed-q341.html.</td>
		</tr>
		<tr>
			<td>Fixation/permeabilization Concentrate</td>
			<td>invitrogen</td>
			<td>Cat#00-5123-43</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixation / Permeabilization Diluent</td>
			<td>invitrogen</td>
			<td>Cat#00-5223-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable Viability Dye EF506</td>
			<td>invitrogen</td>
			<td>Cat#65-0866</td>
			<td></td>
		</tr>
		<tr>
			<td>HiScript II Q RT SuperMix for qPCR (+gDNA wiper)</td>
			<td>Vazyme</td>
			<td>Cat#R223-01</td>
			<td>https://www.vazymebiotech.com/product-center/hiscript-ii-q-rt-supermix-for-qpcr-gdna-wiper-r223.html.</td>
		</tr>
		<tr>
			<td>Leukocyte Activation Cocktail</td>
			<td>BD</td>
			<td>Cat#550583</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-17A (Interleukin 17A) ELISA Kit</td>
			<td>Elabscience&#174;</td>
			<td>Cat#E-EL-M0047</td>
			<td></td>
		</tr>
		<tr>
			<td>Na&#239;ve CD4+ T cells isolation kit, mouse</td>
			<td>STEMCELL</td>
			<td>Cat#19765</td>
			<td>EasySep kit contains mouse CD4+ T cell isolation cocktail [cat no. 19852C.1], mouse memory T cell depletion cocktail [cat no. 18766C], streptavidin RapidSphered 50001 [cat no. 50001], normal rat serum [cat no. 13551]); only store rat serum at -20 &#176;C; other components to be stored at 2-8 &#176;C.</td>
		</tr>
		<tr>
			<td>Permeabilization Buffer</td>
			<td>invitrogen</td>
			<td>Cat#00-8333-56</td>
			<td></td>
		</tr>
		<tr>
			<td>SPARKeasy Cell RNA Kit</td>
			<td>Sparkjade</td>
			<td>Cat#AC0205-B</td>
			<td>https://www.sparkjade.com/product/detail?id=85</td>
		</tr>
		<tr>
			<td>Experimental models: Organisms/strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse: C57BL/6</td>
			<td>Gempharmatech</td>
			<td>Cat#000013</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligonucleotides</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Il17a TaqMan primers with probe</td>
			<td>ribobio</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Il17f TaqMan primers with probe</td>
			<td>ribobio</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Il23r TaqMan primers with probe</td>
			<td>ribobio</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Rora TaqMan primers with probe</td>
			<td>ribobio</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-actin TaqMan primers with probe</td>
			<td>ribobio</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Software and algorithms</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytek Aurora</td>
			<td>Cytek</td>
			<td>https://spectrum.cytekbio.com/</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo v10.8.1</td>
			<td>Tree Star</td>
			<td>https://www.flowjo.com</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad prism 9</td>
			<td>GraphPad Software</td>
			<td>https://www.graphpad-prism.cn</td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Kindly</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Centrifuge tubes</td>
			<td>Eppendorf</td>
			<td>Cat#MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Round-bottom tubes</td>
			<td>Corning</td>
			<td>Cat#352235</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge tubes</td>
			<td>NEST</td>
			<td>Cat#601052</td>
			<td></td>
		</tr>
		<tr>
			<td>48-Well tissue culture plate (flatten bottom)</td>
			<td>Corning</td>
			<td>Cat#3548</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge tubes</td>
			<td>NEST</td>
			<td>Cat#602052</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Cell strainer</td>
			<td>Biosharp</td>
			<td>Cat#BS-70-XBS</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Unskirted qPCR Plates</td>
			<td>VIOX scientific</td>
			<td>Cat#V4801-M</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri dish</td>
			<td>Corning</td>
			<td>Cat#430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5425R</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture CO2 incubator</td>
			<td>Thermo Fisher</td>
			<td>HEPA Class100</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytek Aurora</td>
			<td>Cytek</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>dissecting scissors</td>
			<td>RWD</td>
			<td>S12003-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>AlphaCell</td>
			<td>Cat#J633201</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000 Spectrophotometer</td>
			<td>Thermo Fisher</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-time PCR System</td>
			<td>Roche</td>
			<td>LightCycler96</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tweezers</td>
			<td>RWD</td>
			<td>F12005-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Bio-Rad</td>
			<td>C1000 Touch</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro differentiation of naive cd4+ t cells into pathogenic th17 cells in mouse

After leaving the thymus, naive CD4+ T lymphocytes pass through secondary lymphoid organs. Antigen-presenting cells that transmit homologous antigens to na&#239;ve CD4+ T cells activate them, starting a series of differentiation programs that eventually result in the production of highly specialized T helper (Th) cell lineages1. Interleukin 17 (IL-17) production characterizes Th17 cells, a subpopulation of pro-inflammatory Th cells2. Th17 cells play a role in the host&#39;s defense against extracellular pathogens and in the pathogenesis of many autoimmune diseases, such as autoimmune uveitis and multiple sclerosis. Signals from T-cell receptors and cytokines IL-6 and transforming growth factor-&#946; (TGF-&#946;) induce the differentiation of naive T cells into Th17 cells through the phosphorylation of signal transducer and activator of transcription (STAT)33. STAT3 is further amplified in a positive feedback loop through signaling mediated by IL-23 and IL-214,5. Phosphorylation of STAT3 can induce the expression of the transcription factors ROR&#947;t and ROR&#945;, which act as master switches regulating the cytokine profile of IL-17A, IL-17F, IL-21, and IL-22 in Th17 cells6. However, it has been reported that IL-6- and TGF-&#946;-induced Th17 cells are insufficient to trigger autoimmune diseases, which require co-stimulation by IL-23 or separate co-stimulation of IL-6, IL-1&#946;, and IL-23 in the absence of TGF-&#946;7,8.
Th17 subsets that cannot effectively induce experimental autoimmune encephalomyelitis (EAE) are sometimes referred to as non-pathogenic Th17 while Th17 subsets that can induce EAE are known as pathogenic Th179. Current studies have shown that although pathogenic Th17 and non-pathogenic Th17 co-express the core transcription factor ROR&#947;t, there are great differences in the ability to produce IL-17A and pro-inflammatory and anti-inflammatory properties10. In addition to the high expression of ROR&#947;t, CCR6, STAT4, and RUNX4, which are common characteristic transcription factors of Th17, pathogenic Th17 cells also show additional gene signal expression characteristics related to the disease, such as TBX21, IFN-&#947;, and CXCR3, which have the characteristics of Th1 cell subsets. Pathogenic Th17 cells can secrete high levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-&#947;, TNF-&#945;, and other cytokines11,12. The phenotype of non-pathogenic Th17 cells is unstable, and only under the stimulation of CD3 and cytokine IL-2 can some of these cells differentiate into pathogenic Th17 cells. Therefore, in common clinical disease models such as rheumatoid arthritis, multiple sclerosis, and acute respiratory distress syndrome, pathogenic Th17 cells primarily exert pathogenic effects.
Pathogenic and non-pathogenic Th17 cells can differentiate in vitro under the influence of different cytokines. In recent years, several studies have proposed methods for inducing the differentiation of Th17 cells using different types and concentrations of cytokines. Th17 cells are stimulated by a combination of IL-6, IL-1&#946;, and IL-2313,14,15,16. It has been proved that IL-6 and TGF-&#946;, two cytokines necessary for Th17 cell differentiation, synergistically regulate the expression of ROR&#947;t and Th17 cell differentiation by interacting with two different conserved non-coding DNA sequences at the Rorc gene locus17. The stable phase of pathogenic Th17 cells is mainly maintained by IL-2318,19. IL-23 binds to its receptor and activates the JAK-STAT signaling pathway20, thereby causing phosphorylation of Jak2 and Tyk2 and promoting phosphorylation of STAT1, STAT3, STAT4, and STAT5. IL-4 and IFN-&#947; are negative regulators of this pathway. However, studies have shown that IL-1&#946; may positively regulate the transcription of Ror&#945; and Ror&#947;t through the mTOR pathway to maintain the stability of the Th17 cell phenotype21.
Due to the heterogeneity of numerous studies, we chose the induction protocols for pathogenic and non-pathogenic Th17 cells from the latest research as controls22. The results indicate that, assuming that everything is carried out according to this protocol, after 5 days of culture under the condition of generating pathogenic Th17, more than 90% of the surviving cells can be pathogenic Th17 cells.

<meta charset="utf8"></head><body>Yazar Spotlight: Sitokin Konsantrasyon Modülasyonu Kullanarak Th17 Hücrelerinin Yüksek Saflıkta İn Vitro Farklılaşmasının Elde Edilmesi

Farede Naif CD4+ T Hücrelerinin Patojenik Th17 Hücrelerine İn Vitro Farklılaşması

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

in-vitro-differentiation-naive-cd4-t-cells-into-pathogenic-th17-cells

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Research

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

JoVE Journal

Immunology and Infection

1.2K Görüntüleme.  Southeast University. Araştırma amacımız, Th17 hücrelerinin yönlendirilmiş farklılaşmasını hedeflemektir ve bazı hastalıkla ilişkili Th17 hücreleri ile ilgili yeniden giriş deneyleri için geleneksel bir koşul olarak hizmet eden Th17 hücrelerinin yeniden giriş farklılaşması için basit bir yöntem keşfetmeyi amaçlıyoruz. Birçok çalışma, Th17 hücrelerinin yeniden yerleştirilmesi için yöntemlerden bahsetmiştir ve son araştırmalar küçük molekül...

Video: Yazar Spotlight: Sitokin Konsantrasyon Modülasyonu Kullanarak Th17 Hücrelerinin Yüksek Saflıkta İn Vitro Farklılaşmasının Elde Edilmesi

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.