2019/07/18 14:11
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安捷伦Seahorse XFe24 细胞能量代谢分析仪
型号: Seahorse XFe24
产地: 美国
品牌: 安捷伦
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方案详情:
归一化验证
RAW264.7、HT29 和 SKOV3 细胞在补充了 10% FBS (HyClone,
SH30070.03)、2 mmol/L L-谷氨酰胺 (Corning, 25-005-CI) 和
1 mmol/L 丙酮酸钠 (Corning, 25-000-CI) 的高浓度葡萄糖
DMEM (Gibco, 11960-44) 基础生长培养基中培养。将 A549 细
胞在补充了 10% FBS 和 2 mmol/L GlutaMAX (Gibco, 35050-
061) 的 DMEM:F12 (Corning, 10-090-CV) 基础生长培养基中
培养。将 MCF7 细胞在含有 10% FBS 和 1 mmol/L 丙酮酸钠
的 RPMI-1640 (Gibco, 21870) 中培养。将 HepG2 细胞在补充
了 10% FBS、2 mmol/L GlutaMAX 和 1 mmol/L 丙酮酸的低
浓度葡萄糖 DMEM (Gibco, 11885-084) 中培养。对于 XF96,
以五种不同细胞密度接种细胞,其涵盖范围为建议细胞密度的
±50%;HepG2 为 1 × 104 个细胞/孔,A549、HT29、MCF7 和
SKOV3 为 2 × 104 个细胞/孔,RAW264.7 为 3 × 104 个细胞/
孔。对于 XF24,以三种不同细胞密度接种细胞,涵盖范围为
±25%;HepG2 为 2 × 104 个细胞/孔,A549 和 MCF7 为 4 ×
104 个细胞/孔。
将细胞培养过夜,按照标准安捷伦 Seahorse XF 细胞能量代谢
表型测试方案1 测量代谢表型,其中对方案作出以下修改:将
20 μmol/L Hoechst 33342 (Thermo, 62249) 加入寡霉素/FCCP
混合物中。通过 BioTek Cytation 1/5 使用 XF 细胞成像和计数
软件2 对细胞计数,在 Wave 软件中将数据归一化。使用安捷
伦 Seahorse XF 细胞能量代谢表型报告生成器比较归一化后的
数据变化3。
使用不含酚红的安捷伦 Seahorse XF 基础培养基(安捷伦科
技公司,103335-100)中的细胞进行所有 XF 分析,该培养基
补充了 10 mmol/L 葡萄糖、2 mmol/L L-谷氨酰胺、1 mmol/L
丙酮酸钠和 5 mmol/L HEPES,pH 7.4(安捷伦科技公司,
103337-100)。
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