CD4的制备<sup> +</sup> T细胞通过显微镜分析GD3和GD2神经节苷脂膜表达的
Summary
我们描述了在显微镜使用，以确定在静止神经节苷脂和激活的人幼稚CD4 + T细胞的细胞膜表达和定位一个标准抗体染色方案。还使用<40,000个细胞，不需要额外的低输入RNA试剂描述的实时PCR实验。
Abstract
在悬浮液中和对幼稚CD4 + T细胞的活化本文描述的方法及其在盖玻片共聚焦显微镜分析依从允许参与CD4 + T细胞活化神经节苷脂的空间定位和可视化，即补体表达分析实验，如流式细胞仪，西印迹或实时PCR。神经节苷脂表达通过流式细胞仪，并通过显微镜它们的细胞定位的量化可以通过使用抗神经节苷脂抗体以高亲和力和特异性来获得。然而，在悬浮细胞的适当处理涉及培养板的处理，以促进对荧光或共聚焦显微镜获得所需的必要的粘附性。在这项工作中，我们描述了幼稚的 CD4 + T细胞活化期间确定GD3和GD2神经节苷脂表达并与TCR共定位的协议。此外，真正的-T使用<40,000个细胞IME PCR实验为GD3和GM2 / GD2合酶基因的测定中描述，这表明基因分析实验可以低数量的细胞，无需额外的低输入的RNA试剂盒来进行。
Introduction
的CD4 + T细胞协调通过其效应子功能由抗原呈递细胞1活化后的免疫应答。被激活期间调制的细胞机制的研究允许洞察免疫功能的一基本过程。然而，因为它们在血液周2代表细胞的非常小的群体幼稚CD4 + T细胞的研究可能是复杂的。
通过荧光显微镜几个报告已经研究参与CD4 + T细胞活化的不同分子，主要关联至质膜3蛋白的定位。神经节苷脂含唾液酸鞘糖脂，虽然他们已经在他们丰富，其他细胞等免疫细胞的神经细胞被广泛研究，也表达了与生物学相关职能4,5神经节苷脂。我们以前报道THA人幼稚CD4 + T细胞的活化有所述α2,8唾液酸转移酶ST8Sia 1（GD3合酶）和GM2 / GD2合酶的上调在t，诱导GD2的显著表面neoexpression和GD3神经节苷脂6的上调。在免疫细胞GD3，GD2和其他神经节苷脂的进一步研究是必要的，以补充免疫功能的蛋白质为基础的局部视图。
通常，神经节苷脂表达的研究是基于技术如薄层色谱法（TLC）7，但该技术不允许神经节苷脂的空间定位在质膜或在亚细胞区室，限制生物分析。
在这项工作中，我们描述了在人幼稚CD4 + T细胞和PBMC中抗CD3 /抗CD28活化后GD3和GD2神经节苷脂的抗体介导的识别和定位的协议。有了这个协议，我就也有可能以分析神经节苷脂的基因和分子表达在低数量在悬浮细胞，以获得高质量的图像6，考虑淋巴细胞的小尺寸（9微米）。
Protocol
Representative Results
Discussion
所述协议可以用于本地化神经节苷脂或蛋白质中的 CD4 + T细胞或其它免疫细胞（ 例如 ，PMBCs， 图5）由少数细胞起始的细胞悬浮液。因为T细胞和非粘附性的小尺寸的，收购的信息差或低质量的荧光显微图像的结果，如果该细胞不正确地附着。
该协议具有良好质量的共聚焦显微镜分析相结合是对使用技术如薄层色谱法检测神经节苷脂的重要?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We are grateful to Dr. José Luis Daniotti for comments. We thank the assistance to Dr. J. Arturo Pimentel and the Laboratorio Nacional de Microscopìa Avanzada-UNAM for acquisition of confocal images. I.M.-D. and T.M.V.C. were supported by grants 157634 and 253596 from Consejo Nacional de Ciencia y Tecnologìa (CONACYT), Sociedad Latinoamericana de Glicobiologìa, A.C. T.M.V.-C. is recipient of a scholarship (245192) from CONACYT. We also thank the support of the Red Temática Glicociencia en Salud – CONACYT (253596).
Materials
| Advanced RPMI 1640 | Thermo Fisher Scientific | 12633-012 | Suplemented with 3% FBS |
| mouse anti human CD3 antibody | eBioscience | 16-0037-81 | OKT3 clone |
| mouse anti human CD28 antibody | ebioscience | 16-0288-81 | CD28.6 clone |
| mouse anti human GD3 antibody | Abcam | ab11779 | R24 clone |
| mouse anti human GD2 antibody | Santa Cruz Biotechnology | sc-53831 | 14G2a clone |
| FITC-conjugated anti-mouse IgG3 antibody |
Abcam | ab97259 | |
| Alexa Fluor 488 conjugated antimouse | Thermo Fisher Scieni¡tific | A-21131 | |
| IgG2a antibody | |||
| Alexa Fluor 647 conjugated antimouse | Thermo Fisher Scieni¡tific | A-21241 | |
| IgG2a antibody | |||
| Hoechst 333258 | Sigma-Aldrich | 861405 | |
| Ficoll Paque-Plus | GE | 17-1440-02 | |
| Trizol Reagent | Invitrogen | 15596-026 | |
| Naive CD4+ T Cell Isolation Kit II, human | MACS Miltenyi Biotec | 130-094-131 | |
| BD FACSAria II | BD Biosciences | Sorting | |
| Nunc Lab-tek chamber slide system | Sigma-Aldrich | C7182 | |
| Olympus FV 1000 Laser Confocal Microscope (Olympus) | Olympus | Upright BX61WI and IX81 | |
| Forward sequence primer GD3 synthase | 5´-GAGCGTTCAGGAAACAAATGG- 3´ | Ref. 7 | |
| Reverse sequence primer GD3 synthase | 5´-CCTGTGGGAAGAGAGAGTAAG-3´ | Ref. 7 | |
| Forward sequence primer GM2/GD2 synthase | 5´-CAACACAGCAGACACAGTCC-3´ | Ref. 7 | |
| Reverse sequence primer GM2/GD2 synthase | 5´-GTGGCAATCGTGACTAGAGC-3´ | Ref. 7 |
References
- Mazzon, C., Viola, A. From tango to quadrilla: current views of the immunological synapse. Cell Adh Migr. 1 (1), 7-12 (2007).
- Hamann, D., Pa Baars, ., Hooibrink, B., van Lier, R. W. Heterogeneity of the human CD4+ T-cell population: two distinct CD4+ T-cell subsets characterized by coexpression of CD45RA and CD45RO isoforms. Blood. 88 (9), 3513-3521 (1996).
- Zhong, L., Zhang, Z., Lu, X., Liu, S., Chen, C. Y., Chen, Z. W. NSOM / QD-Based Visualization of GM1 Serving as Platforms for TCR / CD3 Mediated T-Cell Activation. Biomed Res Int. , 276498 (2013).
- Nagafuku, M., Okuyama, K., et al. PNAS Plus: CD4 and CD8 T cells require different membrane gangliosides for activation. Procs Natl Acad Sci USA. 109 (6), E336-E342 (2012).
- Schnaar, R. L., Gerardy-Schahn, R., Hildebrandt, H. Sialic acids in the brain: gangliosides and polysialic acid in nervous system development, stability, disease, and regeneration. Physiol Rev. 94, 461-518 (2014).
- Villanueva-Cabello, T. M., Mollicone, R., Cruz-Muñoz, M. E., Lòpez-Guerrero, D. V., Martìnez-Duncker, I. Activation of human naïve Th cells increases surface expression of GD3 and induces neoexpression of GD2 that colocalize with TCR clusters. Glycobiology. 25 (12), 1454-1464 (2015).
- Müthing, J. TLC in structure and recognition studies of glycosphingolipids. Methods Mol Biol. 76 (17), 183-195 (1998).
- de Almeida, M. C., Silva, A. C., Barral, A., Barral Netto, M. A simple method for human peripheral blood monocyte isolation. Mem Inst Oswaldo Cruz. 95 (2), 221-223 (2000).
- O’Neil-Andersen, N. J., Lawrence, D. A. Differential modulation of surface and intracellular protein expression by T cells after stimulation in the presence of monensin or brefeldin A. Clin Diagn Lab Immunol. 9 (2), 243-250 (2002).
- Mannering, S. I., Zhong, J., Cheers, C. T-cell activation, proliferation and apoptosis in primary Listeria monocytogenes infection. Immunology. 106 (1), 87-95 (2002).
- Marconi, S., Acler, M., et al. Anti-GD2-like IgM autoreactivity in multiple sclerosis patients. Mult Scler. 12 (3), 302-308 (2006).
- Park, J. E., Wu, D. Y., et al. Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T-cell ligand. Immunology. 123 (1), 145-155 (2008).
- Simon, B. M., Malisan, F., Testi, R., Nicotera, P., Leist, M. Disialoganglioside GD3 is released by microglia and induces oligodendrocyte apoptosis. Cell Death Differ. 9 (7), 758-767 (2002).
- Beske, O., Reichelt, M., Taylor, M. P., Kirkegaard, K., Andino, R. Poliovirus infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of the Golgi complex. J Cell Sci. 120 (18), 3207-3218 (2007).
- Zuber, C., Spiro, M. J., Guhl, B., Spiro, R. G., Roth, J. Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control. Mol Biol Cell. 11 (12), 4227-4240 (2000).
- Yamashiro, S., Okada, M., et al. Expression of (GD3 synthase) gene in human cancer cell lines: high level expression in melanomas and up-regulation in activated T lymphocytes. Glycoconj J. 12 (6), 894-900 (1995).
- Wipfler, D., Srinivasan, G. V., et al. Differentially regulated expression of 9-O-acetyl GD3 (CD60b) and 7-O-acetyl-GD3 (CD60c) during differentiation and maturation of human T and B lymphocytes. Glycobiology. 21 (9), 1161-1172 (2011).
- Pukel, C. S., Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., Old, L. J. GD3, a prominent ganglioside of human melanoma. Detection and characterisation by mouse monoclonal antibody. J Exp Med. 155 (4), 1133-1147 (1982).
