Christian J A, Rebar A H, Boon G D, Low P S
Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN 47907-1243, USA.
Exp Hematol. 1996 Jan;24(1):82-8.
Biotinylation of erythrocytes has been developed in rabbits as a tool to retrieve labeled cells following various periods in circulation. This retrieval capability allows biochemical studies to be conducted on red blood cells (RBC) that have aged for desired times in vivo. However, because erythrocyte life span is much shorter in rabbits than in humans, and because cell removal is measurably age-independent in rabbits, we have sought to validate the same protocol in dogs, whose cell life span and age-dependent removal characteristics are similar to humans'. Canine RBC were biotinylated in vivo by infusion of N-hydroxysuccinimidyl biotin dissolved in dimethylacetamide or dimethylsulfoxide. Cell life spans were evaluated using 14C-cyanate labeling followed by scintillation counting or avidin-FITC labeling followed by flow cytometry. Both methods gave identical results. The life span of the biotin-conjugated cells was found to be normal (approximately 110 days), and the stability of the biotin ligand was adequate for efficient retrieval of cells using avidin-coated magnetic beads (magnetic cell sorting [MACS]). From each isolation, approximately 20 microL of packed biotinylated cells of approximately 90% purity (i.e., 10% contamination by unlabeled cells) could be harvested. On average, approximately 60% of the biotinylated cells in any sample could be retrieved. Either multiple isolations or use of larger collection columns will facilitate collection of cell numbers sufficient for biochemical tests. After incorporating several modifications in the previous biotinylation protocol that were required for adaptation to the dog, the methodology can be used to study red cell senescence in an animal that has several pertinent similarities to humans.
红细胞生物素化技术已在兔身上得到应用，作为一种在循环不同时间段后检索标记细胞的工具。这种检索能力使得能够对体内老化至所需时间的红细胞（RBC）进行生化研究。然而，由于兔的红细胞寿命比人类短得多，且兔体内细胞清除与年龄无关，我们试图在狗身上验证相同的方案，因为狗的细胞寿命和年龄依赖性清除特征与人类相似。通过输注溶解在二甲基乙酰胺或二甲基亚砜中的N - 羟基琥珀酰亚胺生物素，在体内对犬红细胞进行生物素化。使用14C - 氰酸盐标记后进行闪烁计数或抗生物素蛋白 - FITC标记后进行流式细胞术评估细胞寿命。两种方法得出的结果相同。发现生物素偶联细胞的寿命正常（约110天），生物素配体的稳定性足以使用抗生物素蛋白包被的磁珠（磁性细胞分选[MACS]）有效检索细胞。每次分离可收获约20微升纯度约为90%（即未标记细胞污染10%）的生物素化细胞沉淀。平均而言，任何样本中约60%的生物素化细胞可被检索到。多次分离或使用更大的收集柱将有助于收集足以进行生化测试的细胞数量。在对先前的生物素化方案进行了几项适应狗所需的修改后，该方法可用于研究与人类有若干相关相似之处的动物的红细胞衰老。
