Effective elimination of fibroblasts in cultures of melanocytes by lowering calcium concentration in TPA depleted medium following geneticin treatment
Effective elimination of fibroblasts in cultures of melanocytes by lowering calcium concentration in TPA depleted medium following geneticin treatment
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DOI：
10.1111/j.1600-0749.1996.tb00089.x
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发表时间：
1996-04-01
期刊：
影响因子：
--
通讯作者：
Morelli, JG
中科院分区：
文献类型：
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作者：
Horikawa, T;Norris, DA;Morelli, JG
黑素细胞在添加佛波酯的培养基中生长良好。然而，佛波酯是代谢稳定的，并对多种细胞反应具有长期影响。不同的研究小组报告说，他们可以培养黑素细胞没有佛波酯。然而，在培养基中没有佛波醇酯污染的成纤维细胞往往难以消除。我们已经开发了一种改进的方法，用于消除成纤维细胞使用遗传霉素在无血清培养基中与bFGF，内皮素-l和无佛波酯。用低浓度的遗传霉素处理有效地消除了污染的成纤维细胞，并且如果遗传霉素最初加入高钙培养基（2 mM）中，随后在除去遗传霉素后立即改变为低钙培养基（0.09mM），则对黑素细胞无害。用这种技术，我们可以很容易地建立纯黑素细胞培养。这种培养方法将为研究黑素细胞在不受佛波酯影响的情况下的细胞反应提供一种有用的工具。
Melanocytes grow well in culture media supplemented with phorbol ester. However, phorbol ester is metabolically stable and has prolonged effects on multiple cellular responses. Different groups have reported that they could cultivate melanocytes without phorbol ester. However, in medium without phorbol esters contaminating fibroblasts are often difficult to eliminate. We have developed an improved method for eliminating fibroblasts using geneticin in serum free medium with bFGF, endothelin-l and no phorbol ester. Treatment with a low concentration of geneticin effectively eliminated contaminating fibroblasts and was not harmful to melanocytes if geneticin was added initially in a high calcium media (2 mM) followed by a change to a low (0.09 mM) calcium media immediately after removal of the geneticin. With this technique we could easily establish pure melanocyte culture. This culture method will provide a useful tool for studies on melanocyte cellular response without the influence of phorbol ester.