Extracellular recovery of methotrexate-polyglutamates following efflux from L1210 leukemia cells.
Extracellular recovery of methotrexate-polyglutamates following efflux from L1210 leukemia cells.
复制标题
L1210 白血病细胞流出后甲氨蝶呤-聚谷氨酸盐的细胞外回收。
DOI：
10.1016/0006-2952(80)90093-3
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发表时间：
1980
影响因子：
5.8
通讯作者：
Chello,PL
中科院分区：
文献类型：
--
作者：
Poser,RG;Sirotnak,F;Chello,PL
方法 ___ C57BL/6 x DBA/2F1 (BD2F1) 小鼠被移植 L1210 小鼠白血病腹水形式，如前所述 [8]。在去除白血病细胞前四小时，给小鼠皮下注射48mg/kg放射性r3H]MTX（Moravek Biochemicals，City of Industry，CA）。 L1210 细胞是从含有 10 mM 葡萄糖的 0' 缓冲盐溶液 [9] 中的腹膜腹水中收获的。将细胞洗涤一次并在相同溶液中稀释至标准吸光度水平（Aso=3）。将一半样品置于 0"(To) 处，其余样品在 37" 处流出 20 分钟 (T+20)。然后将两个样品在0英寸下离心，将上清液部分放在一边，并将细胞重悬至2ml于0英寸0.14M NaCl-0.01M磷酸钠，pH 7.4中。测定等分试样的细胞数量，然后将细胞和上清液级分在 100°C 下加热 15 分钟，以提取药物并灭活细胞缀合酶（羧肽酶）和多聚谷氨酰合成酶。将煮沸的样品离心以除去细胞碎片。将澄清的上清液级分与真实的 MTX 一起上样到 0.9 x 30 cm DEAE-Sephadex 柱，
Methods ___ C57BL/6 x DBA/2F1 (BD2F1) mice were transplanted with the ascitic form of the L1210 murine leukemia as described previously [8]. Four hours prior to removing leukemia cells, mice were injected SC with 48 mg/kg radioactive r3H] MTX (Moravek Biochemicals, City of Industry, CA). L1210 cells were harvested from the peritoneal ascitic fluid in 0'buffered salts solution [9] with 10 mM dextrose. The cells were washed once and diluted in the same solution to a standard absorbance level (Aso= 3). Half the sample was set aside at 0"(To), and the remainder allowed to efflux at 37" for 20 min (T+ 20). Both samples were then centrifuged at 0", the supernatant fraction was set aside, and the cells were resuspended to 2 ml in 0" 0.14 M NaCl-0.01 M sodium phosphate, pH 7.4. An aliquot was assayed for cell number, and then both cells and the supernatant fractions were heated at 100" for 15 min to extract drug and inactivate cellular conjugase (carboxypeptidase) and polyglutamyl synthetase. The boiled samples were centrifuged to remove cellular debris. The cleared supernatant fractions were applied to 0.9 x 30 cm DEAE-Sephadex columns along with authentic MTX,