Sanford J A, Stubblefield E
Somat Cell Mol Genet. 1987 May;13(3):279-84. doi: 10.1007/BF01535210.
We have developed a general technique for making micronucleated cells to use in microcell-mediated chromosome transfer. Growing cells are blocked in mitosis with colcemid, placed in a hypotonic solution for 10 min, and returned to culture medium for 24 h. This treatment promotes the formation of micronuclei within lymphoblast or fibroblast cells. The microcells are generated by cytochalasin B treatment on a Percoll density gradient centrifuged at 43,500g. The resulting mixture of microcells, whole cells, and karyoplasts is filtered through 3-micron pores to obtain a pure microcell preparation. The microcells are fused to recipient whole cells using phytohemagglutinin-P and polyethylene glycol. Advantages of this technique are: donor cells need not be attached to a substrate; and cell lines which form micronuclei in low frequency can still be used efficiently as microcell donors.
我们已经开发出一种用于制造微核细胞以用于微细胞介导的染色体转移的通用技术。将生长中的细胞用秋水仙酰胺阻断在有丝分裂期，置于低渗溶液中10分钟，然后放回培养基中培养24小时。这种处理促进了淋巴母细胞或成纤维细胞内微核的形成。通过在43,500g下离心的Percoll密度梯度上用细胞松弛素B处理来产生微细胞。将所得的微细胞、全细胞和核体混合物通过3微米的孔进行过滤，以获得纯的微细胞制剂。使用植物血凝素-P和聚乙二醇将微细胞与受体全细胞融合。该技术的优点是：供体细胞无需附着于基质；并且在低频下形成微核的细胞系仍可有效地用作微细胞供体。
