STUDIES OF MACROPHAGE COMPLEMENT RECEPTOR - ALTERATION OF RECEPTOR FUNCTION UPON MACROPHAGE ACTIVATION
STUDIES OF MACROPHAGE COMPLEMENT RECEPTOR - ALTERATION OF RECEPTOR FUNCTION UPON MACROPHAGE ACTIVATION
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DOI：
10.1084/jem.141.6.1278
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发表时间：
1975-01-01
影响因子：
15.3
通讯作者：
SILVERSTEIN, SC
中科院分区：
文献类型：
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作者：
BIANCO, C;GRIFFIN, FM;SILVERSTEIN, SC
材料与方法小鼠红细胞和腹膜巨噬细胞获自在纽约洛克菲勒大学饲养的瑞士小鼠，C5缺陷小鼠血清由AKR小鼠的C5缺陷品系（杰克逊实验室，巴尔港，缅因州）制备。红细胞将在Alsever溶液（Animal Blood Center，锡拉丘兹，NY）中的绵羊红细胞（E）在PD（Dulbecco磷酸盐缓冲盐水（PBS）的溶液”a”（15））中洗涤三次，并悬浮在培养基199（Microbiological Associates，Bethesda，MD）中。或含Ca++、Mg++和0.1%明胶（VBG）的Veronal缓冲葡萄糖中（16）。巨噬细胞激活剂。Brewer巯基乙酸盐培养基得自Difco Laboratories，Detroit，Mich.，和4.05%水溶液，根据制造商的说明书制备。巨噬细胞收获和维持小鼠腹腔巨噬细胞的方法是Cohn和Benson（17）的方法，如先前修改的（18）。将细胞在含有20%热去补体（56 ℃，30分钟）胎牛血清的培养基199（Grand Island Biological Co.，Grand Island，NY）、100 U/ml青霉素和100 μ g/ml链霉素。在一些实验中，在收获小鼠的腹膜巨噬细胞之前4天，向小鼠腹膜内注射Iml的Brewer巯基乙酸盐培养基。这些动物的巨噬细胞被指定为活化的。乳胶珠，直径1.1k（Dow Chemical Co.，密歇根州米德兰）在PD中洗涤三次，以6 × 105颗粒/ml的浓度悬浮在PD中，并在4 ℃下储存。Sera.用10'小鼠红细胞在4 ℃吸附5 ml正常兔血清2 h。将吸附的血清在-70 ℃下冷冻并用作细胞毒性测定的补体来源。从C5缺陷型AKR小鼠制备血清，并立即以0.2ml等分试样储存在-70 ℃.在涉及使用补体包被E的所有实验中，它作为C1423的来源。
Materials and MethodsAnimals. Mouse erythrocytes and peritoneal macrophages were obtained from Swiss mice maintained at The Rockefeller University, NY C5-deficient mouse serum was prepared from a C5-deficient strain of AKR mice (Jackson Laboratories, Bar Harbor, Maine). Erythrocytes. Sheep erythrocytes (E) in Alsever's solution (Animal Blood Center, Syracuse, NY) were washed three times in PD (solution" a" of Dulbecco's phosphate-buffered saline (PBS)(15)] and suspended in medium 199 (Microbiological Associates, Bethesda, Md.) or in Veronal-buffered glucose containing Ca++, Mg++, and 0.1% gelatin (VBG)(16). Macrophage activator. Brewer thioglycollate medium was obtained from Difco Laboratories, Detroit, Mich., and a 4.05% aqueous solution prepared according to the manufacturer's instructions. Macrophages. The methods for harvesting and maintaining mouse peritoneal macrophages were those of Cohn and Benson (17), as previously modified (18). Cells were cultivated for 24-48 h in medium 199 with 20% heat-decomplemented (56 C, 30 min) fetal bovine serum (Grand Island Biological Co., Grand Island, NY), 100 U/ml of penicillin, and 100 gg/ml of streptomycin. In some experiments, mice were injected intraperitoneally with 1 ml of Brewer thioglycollate medium 4days before their peritoneal macrophages were harvested. Macrophages from these animals are designated activated. Latex beads, 1.1 k in diameter (Dow Chemical Co., Midland, Mich.) were washed three times in PD, suspended in PD at a concentration of 6 x 10 particles/ml, and stored at 4 C. Sera. 5 ml of normal rabbit serum was adsorbed at 4 C for 2 h with 10'mouse erythrocytes. The adsorbed serum was frozen at-70 C and used as a source of complement for cytotoxic assays. Serum was prepared from C5-deficient AKR mice and stored immediately in 0.2 ml aliquots at-70 C. It served as a source of C1423 in all experiments involving the use of complement-coated E.