Immunocytochemical Profiling of Cultured Mouse Primary Retinal Cells.
Immunocytochemical Profiling of Cultured Mouse Primary Retinal Cells.
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DOI：
10.1369/0022155416689675
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发表时间：
2017-04
期刊：
影响因子：
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通讯作者：
Englund-Johansson U
中科院分区：
文献类型：
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作者：
Zalis MC;Johansson S;Englund-Johansson U
原代视网膜细胞培养和免疫细胞化学是眼科研究的重要实验平台。视网膜细胞从其天然环境到体外环境的翻译导致细胞应激，从而危及其体内表型特征。此外，许多视网膜免疫化学标记物的特异性和稳定性在视网膜细胞培养物中评价不佳。因此，我们在此评估了17种视网膜标志物的表达谱，即恢复素、视紫红质、抑制蛋白、Chx 10、PKC、DCX、CRALBP、GS、波形蛋白、TPRV 4、RBPMS、Brn 3a、β-微管蛋白III、NeuN、MAP 2、GFAP和突触素。在培养的第7天和第18天，将小鼠出生后视网膜细胞的标志物表达谱与其年龄匹配的体内视网膜进行比较。我们证明了所有标记物的体外稳定表达，除了抑制蛋白和CRALBP。随着培养时间的推移以及与年龄匹配的视网膜相比，观察到细胞表达和一些标记物位置的差异。我们假设这些差异可能是文化条件依赖。综上所述，我们建议在将体外结果外推到体内环境之前，对特定培养环境中的抗体进行全面评估。此外，特定细胞类型的鉴定可能需要表达的不同基因或具有结构信息的标记物的组合。
Primary retinal cell cultures and immunocytochemistry are important experimental platforms in ophthalmic research. Translation of retinal cells from their native environment to the in vitro milieu leads to cellular stress, jeopardizing their in vivo phenotype features. Moreover, the specificity and stability of many retinal immunochemical markers are poorly evaluated in retinal cell cultures. Hence, we here evaluated the expression profile of 17 retinal markers, that is, recoverin, rhodopsin, arrestin, Chx10, PKC, DCX, CRALBP, GS, vimentin, TPRV4, RBPMS, Brn3a, β-tubulin III, NeuN, MAP2, GFAP, and synaptophysin. At 7 and 18 days of culture, the marker expression profiles of mouse postnatal retinal cells were compared with their age-matched in vivo retinas. We demonstrate stable in vitro expression of all markers, except for arrestin and CRALBP. Differences in cellular expression and location of some markers were observed, both over time in culture and compared with the age-matched retina. We hypothesize that these differences are likely culture condition dependent. Taken together, we suggest a thorough evaluation of the antibodies in specific culture settings, before extrapolating the in vitro results to an in vivo setting. Moreover, the identification of specific cell types may require a combination of different genes expressed or markers with structural information.