{"id":18126,"date":"2018-11-11T13:00:17","date_gmt":"2018-11-11T05:00:17","guid":{"rendered":"http:\/\/biopioneer.com.tw\/?p=18126"},"modified":"2022-07-20T15:18:48","modified_gmt":"2022-07-20T07:18:48","slug":"mutudc1940%e6%b0%b8%e7%94%9f%e5%8c%96%e5%b0%8f%e9%bc%a0%e6%a8%b9%e7%aa%81%e7%8b%80%e7%b4%b0%e8%83%9e-immortalized-mouse-dendritic-cells%e8%b2%a8%e8%99%9ft0528","status":"publish","type":"post","link":"http:\/\/biopioneer.com.tw\/?p=18126","title":{"rendered":"MutuDC1940\u6c38\u751f\u5316\u5c0f\u9f20\u6a39\u7a81\u72c0\u7d30\u80de | Immortalized Mouse Dendritic Cells\u8ca8\u865fT0528"},"content":{"rendered":"

A<\/strong>bmgood<\/strong>\u4e0d<\/strong>\u673d<\/strong>\u5316\u7cfb\u5217\u5546\u54c1<\/strong> (immortalized cell)-Immortalized Mouse Dendritic Cells (MutuDC1940)<\/strong>\"\"<\/p>\n

\"\"<\/p>\n

\u6c38\u751f\u5316\u5c0f\u9f20\u6a39\u7a81\u72c0\u7d30\u80de\uff08MutuDC1940\uff09| \u8ca8\u865fT0528<\/strong><\/a><\/span><\/p>\n

Immortalized Mouse Dendritic Cells (MutuDC1940)<\/p>\n

\"\"<\/p>\n

\u91ce\u751f\u578bMutuDC1940\u7d30\u80de\u682a\u6e90\u81eaCD11c\uff1aSV40LgT\u8f49\u57fa\u56e0\u5c0f\u9f20\u7684\u5e38\u898f\u6a39\u7a81\u7d30\u80de\u7684\u6c38\u751f\u5316\u813eCD8\u03b1+\u4e9e\u7d44\uff08CD11chigh\uff0cB220-\uff0cDEC205 +\uff0cCD24high\uff0cCD11b-\uff09\u3002MutuDC1940\u7d30\u80de\u901a\u904eup-regulation\u53caco-stimulatory CD40\uff0cCD80\u548cCD86\u5206\u5b50\u4fdd\u7559\u5c0dTLR Ligands\u5982CpG\uff08TLR9-L\uff09\u548cPolyIC\uff08TLR3-L\uff09\u7684\u53cd\u61c9\uff0c\u53calesser extent LPS (TLR4-L)\u523a\u6fc0\u3002\u9664\u4e86\u97ff\u61c9PAMP\u4e4b\u523a\u6fc0\u548c\u7522\u751fTh1\u7d30\u80de\u56e0\u5b50\u5982IL-12\u5916\uff0c\u9019\u4e9b\u7d30\u80de\u9084\u80fd\u5920\u5728MHC-I\u548cMHC-II\u4e0bpresenting antigen\uff0c\u5305\u62ec\u76f4\u63a5antigen presentation\u6297\u539f\u5448\u905e\u548c\u7d30\u80decross-presentation\u3002TLR3 knockout\uff08\u76ee\u9304\u865fT3034\uff09\uff0cTLR9 knockout\uff08\u76ee\u9304\u865fT3035\uff09\u548cIfnar1 knockout\uff08\u76ee\u9304\u865fT3036\uff09MutuDC\u6c38\u751f\u5316\u5c0f\u9f20\u6a39\u7a81\u72c0\u7d30\u80de\uff0c\u9019\u4e9b\u6a39\u7a81\u72c0\u7d30\u80de\u7cfb\u5728vaccine science\u75ab\u82d7\u79d1\u5b78\u548cimmunotherapy\u514d\u75ab\u7642\u6cd5\u4e2d\u63d0\u4f9b\u4e86\u5f37\u5927\u7684\u5de5\u5177\uff0c\u7279\u5225\u662fCD8\u03b1+\u4e9e\u7fa4target antigens\u3002<\/p>\n

\"\"<\/p>\n

\"\"MutuDC1940<\/strong>\u6c38\u751f\u5316\u5c0f\u9f20\u6a39\u7a81\u72c0\u7d30\u80de-: <\/strong>\u7d30\u80de\u7279\u6027: Adherent | \u4e0d\u6747\u65b9\u6cd5: Isolated from C57BL\/6 transgenic mice carrying SV40 Large T oncogene | \u4f86\u6e90CD11c:SV40LgT-transgenic C57BL\/6 mice\u3002\u5668\u5b98\u4f86\u6e90\u00a0\u00a0 Spleen\u3002Cell marker: CD11c, CD24, MHC-II+, DEC205, Clec9A, B220, CD11b, IRF4, IRF8, CD4\u3002\u7d30\u80de\u57f9\u990a\u689d\u4ef6: \u8a72\u7d30\u80de\u7cfb\u7684\u57fa\u790e\u57f9\u990a\u57fa\u662fIMDM\uff081x\uff09+ GlutamaxTM\uff08Gibco Ref\uff1a31980-022\uff09\u3002 \u88fd\u5099\u5b8c\u6210\u7684\u751f\u9577\u57f9\u990a\u57fa\uff0c\u5c07\u4ee5\u4e0b\u7d44\u5206\u6dfb\u52a0\u5230\u57fa\u790e\u57f9\u990a\u57fa\u4e2d\uff1a\u5f9ePAN\u751f\u7269\u6280\u8853\uff08P40-37500\uff09\u6216Nucleus Biologics\uff08\u5373FBS1824-001\uff09\uff0c1\uff05\u76847.5\uff05\u7684\u53bb\u88dc\u9ad4\u80ce\u725b\u8840\u6e05\u81f3\u7d42\u6fc3\u5ea610\uff05 \uff05\u78b3\u9178\u6c2b\u9209\u6eb6\u6db2\uff0c50\u03bcM\u03b2-\u5df0\u57fa\u4e59\u9187\uff0cHEPES\u81f3\u7d42\u6fc3\u5ea6\u70ba1\uff05\uff0c\u9752\u9ef4\u7d20\/\u93c8\u9ef4\u7d20\u6eb6\u6db2\uff08G255\uff09\u81f3\u7d42\u6fc3\u5ea6\u70ba1\uff05\u3002 \u4f7f\u7528\u524d0.22\u03bcm\u904e\u6ffe\u3002Quality Control (<\/strong>1\uff09\u901a\u904eMHC-I\uff08SIINFEKL \/ OT-I\uff09\u548cMHC-II\uff08OVA323-339 \/ OT-II\uff09\u9650\u5236\u7cfb\u7d71\u8a55\u4f30\u76f4\u63a5\u6297\u539f\u5448\u905e\u548c\u4ea4\u53c9\u6297\u539f\u5448\u905e\u3002(2\uff09 \u901a\u904eRT-PCR\u8a55\u4f30proteome profile\u548c\u8868\u9762\u6a19\u8a8c\u7269\u3002(3\uff09\u4f7f\u7528ELISA\u5206\u6790IL-12\u7d30\u80de\u56e0\u5b50\u5206\u6ccc \u3002(4\uff09\u901a\u904e\u529f\u80fd\u6e2c\u5b9a\u8a55\u4f30\u5c0dPAMP\u523a\u6fc0\u7684\u53cd\u61c9\u3002<\/p>\n

\"\"<\/p>\n

T0528\u5f15\u7528\u6587\u737b<\/strong><\/p>\n

1.Fuertes Marraco, SA\u00a0et al. “Novel murine dendritic cell lines: a powerful auxiliary tool for dendritic cell research” Front Immunol 3:331 (2012).\u00a0DOI: 10.3389\/fimmu.2012.00331.<\/p>\n

2. Duval, A\u00a0et al. “Large T Antigen-Specific Cytotoxic T Cells Protect Against Dendritic Cell Tumors through Perforin-Mediated Mechanisms Independent of CD4 T Cell Help” Front Immunol 5:338 (2014).\u00a0DOI: 10.3389\/fimmu.2014.00338.<\/p>\n

3. Marraco, SA\u00a0et al. “Novel Murine Dendritic Cell Lines: A Powerful Auxiliary Tool for Dendritic Cell Research” Front Immunol 3:331 (2012).\u00a0DOI: 10.3389\/fimmu.2012.00331.<\/p>\n

4. Fuertes Marraco , SA\u00a0et al. “Novel murine dendritic cell lines: a powerful auxiliary tool for dendritic cell research” Front Immunol. 3:331 (2012).\u00a0PubMed: 23162549.<\/p>\n

5. Joshi, PS\u00a0et al. “Characterization of immortalized human mammary epithelial cell line HMEC 2.6” Tumour Biol. 39(10):1010428317724283 (2017).\u00a0DOI: 10.1177\/1010428317724283.\u00a0PubMed: 29022488.<\/p>\n

\n

Decomplement<\/strong>\u52a0\u71b1\u975e\u6d3b\u5316<\/strong>FBS (<\/strong>heat-inactivate FBS)\uff1a<\/strong><\/p>\n

1.\u8b93FBS\u74f6\u57284\u00b0C\u4e0b\u5b8c\u5168\u89e3\u51cd\u904e\u591c\u3002<\/p>\n

2.\u5c07FBS\u74f6\u7f6e\u65bc56\u00b0C\u7684\u6c34\u6d74\u4e2d30\u5206\u9418\u3002<\/p>\n

Decomplemented FBS (Heat-inactivated FBS) \u53ef\u4ee5\u5728-20\u00b0C\u4e0b\u9577\u671f\u4fdd\u5b58\u3002\u907f\u514d\u983b\u7e41\u7684\u51cd\u878d\u5faa\u74b0\u3002\u6bcf2-3\u5929\u66f4\u63db\u4e00\u6b21\u5b8c\u6574\u7684\u5a92\u9ad4\u3002\u4e0d\u8981\u8b93\u4ecb\u8cea\u984f\u8272\u8b8a\u70ba\u9ec3\u8272\u3002\"\"<\/p>\n

Thawing\u7d30\u80de<\/strong>\u89e3\u51cd<\/strong>\u65b9\u6cd5<\/strong>\uff1a<\/strong>(<\/strong>\u6bcf\u7a2e\u7d30\u80de\u5efa\u8b70\u689d\u4ef6\u53ef\u80fd\u6709\u8a31\u4e0d\u540c\uff0c\u4f9d\u7167\u539f\u5ee0\u5efa\u8b70\u64cd\u4f5c\uff0c\u4ee5\u4e0b\u70ba\u901a\u7528\u65b9\u5f0f<\/strong>)<\/strong><\/p>\n

1.\u572837\u2103\u6c34\u6d74\u4e2d\u89e3\u51cd\u5c0f\u74f6\uff0c\u76f4\u5230\u53ea\u6709\u4e00\u5c0f\u584a\u51b0\u584a\u3002<\/p>\n

2.\u5c07\u7d30\u80de\u8f49\u79fb\u5230\u542b\u67095ml pre-warmed complete media \u4e4b15ml\u7ba1\u4e2d\u3002<\/p>\n

3.\u5c07\u7d30\u80de\u4ee5290xg\u96e2\u5fc35\u5206\u9418\u3002<\/p>\n

4.\u5c0f\u5fc3\u4e1f\u68c4\u4e0a\u6e05\u6db2\uff0c\u4e0d\u8981\u6253\u64fe\u7d30\u80de\u6c89\u6fb1\uff0c\u8f15\u8f15\u5730\u4e0a\u4e0b\u79fb\u6db2\uff0c\u5c07\u7d30\u80de\u91cd\u65b0\u61f8\u6d6e\u57281ml\u5b8c\u5168\u57f9\u990a\u57fa\u4e2d\u3002<\/p>\n

5.\u5c07\u7d30\u80de\u57f9\u990a\u572820,000 – 60,000 cells<\/span>\u500b\u7d30\u80de\/ cm2\u3002<\/p>\n

 <\/p>\n

Subculture<\/strong>\u7d30\u80de\u7e7c\u4ee3\u65b9\u6cd5<\/strong>:<\/strong> (<\/strong>\u6bcf\u7a2e\u7d30\u80de\u5efa\u8b70\u689d\u4ef6\u53ef\u80fd\u6709\u8a31\u4e0d\u540c\uff0c\u4f9d\u7167\u539f\u5ee0\u5efa\u8b70\u64cd\u4f5c\uff0c\u4ee5\u4e0b\u70ba\u901a\u7528\u65b9\u5f0f<\/strong>)<\/strong><\/p>\n

1.\u7576\u7d30\u80de70-90\uff05 confluency\u6642\uff0c\u5efa\u8b70\u7e7c\u4ee3\u57f9\u990a\u3002<\/p>\n

2. Aspirate old media\u3002\u61f8\u6d6e\u6db2\u4e2d\u7684\u4e00\u4e9b\u7d30\u80de\u4ecd\u7136\u662f\u6d3b\u7d30\u80de\u3002\u6216\u8005\uff0c\u4e5f\u53ef\u4ee5\u6536\u96c6\u542b\u6709\u61f8\u6d6e\u7d30\u80de\u7684\u4e0a\u6e05\u6db2\u4e26\u96e2\u5fc3\uff08Skip to Step 6 in protocol\uff09\u3002<\/p>\n

3.\u52a0\u51651\uff1a1\u6bd4\u4f8b\u76841X\u7121\u83ccPBS\u548c0.25\uff05\u80f0\u86cb\u767d\u9176-EDTA(TM051<\/a>)\u3002<\/p>\n

4.\u5728\u5ba4\u6eab\u4e0b\u5b75\u80b2\u7d30\u80de3-5\u5206\u9418\u4e26\u652a\u62cc\u57f9\u990a\u5bb9\u5668\u76f4\u81f390\uff05\u7684\u7d30\u80de\u812b\u843d\u3002<\/p>\n

5. \u4e2d\u548c\u80f0\u86cb\u767d\u9176:\u6dfb\u52a0\u7b49\u91cf\u4e4btrypsin + PBS\u3002<\/p>\n

6.\u6536\u96c6\u7d30\u80de\u4e26\u4ee5290xg\u96e2\u5fc35\u5206\u9418\u3002<\/p>\n

7.\u4e1f\u68c4\u4e0a\u6e05\u6db2\uff0c\u8f15\u8f15\u5730\u4e0a\u4e0b\u79fb\u6db2\uff0c\u5c07\u7d30\u80de\u91cd\u65b0\u61f8\u6d6e\u5728\u5b8c\u5168\u57f9\u990a\u57fa\u4e2d\u3002<\/p>\n

8.\u5efa\u8b70split ratio\u6bd4\u4e0d\u8d85\u904e1\uff1a3\u3002<\/p>\n

\u53ef\u4ee5\u4f7f\u7528PolyIC\uff085\u03bcg\/ ml\uff09\uff0cCpG\uff082mM\uff09\u6216LPS\uff085\u03bcg\/ ml\uff09\u9032\u884c\u523a\u6fc0\u3002\u9019\u4e9b\u7d30\u80de\u5c0dFBS\u7684\u8981\u6c42\u7279\u5225\u654f\u611f\uff0c\u56e0\u6b64\uff0c\u5efa\u8b70\u4f7f\u7528\u76f8\u540c\u7684\u6279\u6b21\u57f9\u990a\u80fd\u5920\u652f\u6301\u5176\u57f9\u990a\u7684\u6700\u4f73\u7d50\u679c\u7684\u7d30\u80de\u3002\u6211\u5011\u9084\u5efa\u8b70\u5728\u5b8c\u6574\u57f9\u990a\u57fa\u4e2d\u6dfb\u52a0\u984d\u5916\u76841\uff05Hepes\u4ee5\u4fc3\u9032\u7d30\u80de\u7e41\u6b96\u3002<\/p>\n

Freezing\u7d30\u80de<\/strong>\u51cd\u7d50\uff1a<\/strong>(<\/strong>\u6bcf\u7a2e\u7d30\u80de\u5efa\u8b70\u689d\u4ef6\u53ef\u80fd\u6709\u8a31\u4e0d\u540c\uff0c\u4f9d\u7167\u539f\u5ee0\u5efa\u8b70\u64cd\u4f5c\uff0c\u4ee5\u4e0b\u70ba\u901a\u7528\u65b9\u5f0f<\/strong>)<\/strong><\/p>\n

Complete growth medium with decomplemented FBS to a final concentration of 50% from PAN Biotech (P40-37500) or from Nucleus Biologics (FBS1824-001) and 5% DMSO.<\/p>\n

\u5132\u5b58\u6eab\u5ea6\uff1a\u6db2\u6c2e\u6c23\u76f8\u3002\u91cd\u8981\u63d0\u793a\uff1a\u6b64\u7d30\u80de\u4fc2\u5c0dFBS\u6279\u6b21\u975e\u5e38\u654f\u611f\u3002\u6211\u5011\u5f37\u70c8\u5efa\u8b70\u6700\u7d42\u7528\u6236\u4f7f\u7528\u5f9ePAN biotech\u6216Nucleus Biologics \u4e4bFBS\u3002<\/p>\n

QC\u6aa2\u6e2c:<\/p>\n

1) Direct antigen presentation and cross-antigen presentation were evaluated by the MHC-I (SIINFEKL\/OT-I ) and MHC-II (OVA323-339\/OT-II) restricted systems; 3) proteome profile and surface markers assessed by RT-PCR; RT-PCR; 3) IL-12 cytokine secretion analyzed using ELISA ; 4) Response to PAMP stimulation evaluated by functional assays<\/p>\n

T0528\u5f15\u7528\u6587\u737b<\/strong><\/p>\n