Okada T, Metcalfe D D, Ito Y
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md., USA.
Int Arch Allergy Immunol. 1996 Apr;109(4):376-82. doi: 10.1159/000237266.
A method for cell purification was designed without using high-density media which may impair membrane receptors. Rat and mouse mast cells were separated with an improved nonsynchronous flow-through coil planet centrifuge. Peritoneal cells were suspended at a concentration of 2-3x10(7) cells/ml in conditioned RPMI 1640, supplemented with 50% heat-inactivated FCS and 0.32% sodium citrate. In each separation 3 ml of cell suspension were loaded into the coiled column and elutriated at 4 degrees C. Several conditions, including the centrifugal force, revolution/rotation ratio, density of separation media, flow speed, and designs of both coiled column and flow tubes, were examined and optimized for mast cell purification. Rat mast cells were separated at the purity of 99.2%, with an average yield of 40% under sterile conditions. Nearly 90% pure mouse mast cells were harvested, despite a very low population of mast cells available in murine peritoneal cells. Purified cells were morphologically intact and discharged granules by exocytosis as indicated by electron-microscopic observations. The average histamine release with antigenic specificity was 34 and 61%, in passive sensitization in vitro and in vivo, respectively. Mast cells sensitized with mouse monoclonal IgE antibody released histamine, similar to cells sensitized with homologous antibody. This newly devised method of cell separation will be useful to purify biologically intact mast cells.
设计了一种不使用可能损害膜受体的高密度介质的细胞纯化方法。使用改进的非同步流通盘管行星离心机分离大鼠和小鼠肥大细胞。将腹膜细胞以2-3×10⁷个细胞/毫升的浓度悬浮于条件RPMI 1640中，补充50%热灭活胎牛血清和0.32%柠檬酸钠。每次分离时，将3毫升细胞悬液装入盘管柱中，并在4℃下淘洗。研究并优化了包括离心力、转/转速比、分离介质密度、流速以及盘管柱和流管设计在内的多个条件以用于肥大细胞纯化。在无菌条件下，大鼠肥大细胞的分离纯度为99.2%，平均产量为40%。尽管小鼠腹膜细胞中肥大细胞的数量非常少，但仍收获了近90%纯度的小鼠肥大细胞。电子显微镜观察表明，纯化后的细胞形态完整，并通过胞吐作用排出颗粒。在体外和体内被动致敏中，具有抗原特异性的组胺平均释放率分别为34%和61%。用小鼠单克隆IgE抗体致敏的肥大细胞释放组胺，类似于用同源抗体致敏的细胞。这种新设计的细胞分离方法将有助于纯化生物学上完整的肥大细胞。
