PMEFFEEDERLAYERCONCENTRATION
Area/WellTotal AreaTotal CellsTotal VolConc/mlFlask cm2/cm25x104/cm296 Well0.35374.62x10519.52.4X10424 Well1.7742.45.3x105252.1X10412 Well3.541.65.2x105251.9X1046 Well9.657.67.0x105371.9X104Large F11541541.9x106209.5X104Med F1~175801x106101X105Small F1~3024.43x10556X104Large Dish1431.8x106209X104Med Dish570.7x10107x104Small Dish9.61.2 X 10526X104......阅读全文
PMEF－FEEDER－LAYER－CONCENTRATION
Area/WellTotal AreaTotal CellsTotal VolConc/mlFlask cm2/cm25x104/cm296 Well0.35374.62x10519.52.4X10424 Well1.7742.45.3x105252.1X10412 Well3.541.65.2x1
Routine－Culturing－of－ES－Cells
Cell are normally passaged every 2-3 days, this is important to avoid differentiation.Signs of differentiation are:-i) colonies are surrounded by flat
胚胎干细胞培养
Media and Solution required for ES Cell Culture (Bowtell Lab) Routine Culturing of ES Cells (Bowtell Lab) Routine Splitting and freezing of cells (
Gamma－Irradiation－of－PMEFs
Thawing Cells-Remove a vial of Passage 2 Primary Mouse Embryo Fibroblasts (PMEF'S), at a concentration of 3 x 106 from liquid nitrogen and thaw qu
胚胎干细胞培养技术大全
MEDIA AND SOLUTIONS REQUIRED FOR ROUTINE ES CELL CULTURERoutine Culturing of ES CellsISOLATION OF PRIMARY MOUSE EMBRYO FIBROBLASTSMITOMYCIN C TREATMEN
Antibiotic－Concentration－in－Media
Antibiotics should be added to lower than 60 C broth, or filter sterilized: See note. "C'' refers to chromosomal resistance: "P" plasmid based
Feeder－Pathways－for－Glycolysis
The glycolytic pathway begins with the simple sugar glucose and leads to pyruvate and eventually the Kreb's cycle. Dietary carbohydrates include a
Bradford－Protein－Concentration－Assay
Bradford Protein Concentration Assayversion 01/07/2001Abbreviations:mcg = microgramsmcL = microlitersBSA = bovine serum albuminO.D. = optical densityd
小鼠feeder细胞分离
You will need:13.5 day pregnant mouse (we use MTK NEO inbred white mice)2 sets sterile instrumentsone containing a pair of curved forceps and a pair o
Thin－Layer－Chromatography
Reaction volume 100 ul5 ul NADPH (100 mM)x ul cell extract so you get 50 % conversion0.2 uM C14-Testosteroneadd Tris-Citrate Buffer pH 6 to an endvolu
Protein－concentration－of－Laemmli－gel－samples
Protein concentration of Laemmli gel samplesTo 10 µl boiled lysate (in Laemmli sample buffer) add 40µl water + 50µl 50% TCA. Ppt. 10 min. on ice. Spin
FeederFree－Culture－of－hESCs
MEF Conditioned mediumPlate MEFs (p4 or p5) onto gelatin-coated plates at a density of 1x106 cells/10cm culture dish.The next day, wash cells with PBS
使用CCCadvanced™FN1无异源耗材培养人多能干细胞（一）
Ready-to-use Eppendorf CCCadvanced™ FN1 Motifs Surface for Xeno-Free Expansionof Human Pluripotent Stem CellsAurélie Tacheny¹, Silvia Tejerina¹, Wiâme
使用饲细胞(Feeder－Cells)制备
（1）取原代培养的人或动物胚胎成纤维细胞，90%汇合时制成细胞悬液，再按105细胞/毫升重新接种培养。（2）在细胞半汇合时，准备0.25μg/ml的丝裂霉素C，按2μg/106细胞的量加入到培养瓶中过夜；或用射线单次照射，剂量30～50戈瑞。（3）细胞经上述处理后，Hanks液漂洗两次、更换培养液、
蛋白浓缩(protein－concentration)方法详解
1，透析袋浓缩法利用透析袋浓缩蛋白质溶液是应用最广的一种。将要浓缩的蛋白溶液放入透析袋（无透析袋可用玻璃纸代替），结扎，把高分子（6000－12000）聚合物如聚乙二醇（碳蜡）、聚乙烯吡咯、烷酮等或蔗糖撒在透析袋外即可。也可将吸水剂配成30%－40%浓度的溶液，将装有蛋白液的透析袋放入即可。吸水剂用
蛋白浓缩（protein－concentration）基本方法
蛋白浓缩方法基本有：丙酮沉淀法；免疫沉淀法；三氯醋酸沉淀法；硫酸铵沉淀法；（低温）有机溶剂沉淀法；聚乙二醇沉淀法；超滤法；透析法；离子交换层析和冷冻干燥法…… 1.丙酮沉淀法；三氯醋酸沉淀法 试验要求的仪器简单，但是常常导致蛋白质变性。 2.免疫沉淀法：得有特异性抗体！ 3.硫酸铵沉淀法
Immunostaining－Thin－Layer－Chromatograms－Of－Glycolipids
Immunostaining Thin Layer Chromatograms Of GlycolipidsJohn L. Magnani~GlycoTech Corporation, Rockville, Maryland 20850Immunostaining thin layer chroma
stem－cell－culture－protocol
实验概要stem cell culture protocol主要试剂cell culture supplies and reagentssEnvironment: cell culture requires a sterile environment, so it needs a separat
Growing－feederindependent－embryonic－stem－cells§
We use feeder-independent ES cell lines derived from the 129/Ola strain of mice (Nichols et al., Development 110, p.1341, 1990). These cells are easy
层析技术(Layeranalise－technique)
离子交换层析技术是以离子交换纤维素或以离子交换葡聚糖凝胶为固定相，以蛋白质等样品为移动相，分离和提纯蛋白质、核酸、酶、激素和多糖等的一项技术。（一）原理在纤维素与葡聚糖分子上结合有一定的离子基团，当结合阳离子基团时，可换出阴离子，则称为阴离子交换剂。如二乙氨乙基（Dicthylaminoethyl，
Procedure－for－Culturing－BG01V－Human－Embryonic－Stem－Cells
IntroductionHuman embryonic stem (hES) cells are pluripotent stem cells derived from pre-implantation embryos that can be maintained and expanded in a
Multicolour－3DFISH－in－vertebrate－cells4
DetectionThe choice of the detection scheme depends on several factors: (i) the number of haptens and fluorochromes used for probe labeling; (ii) the
薄层层析(thin－layer－chromatography)操作要点
铺板铺板用的匀浆不宜过稠或过稀：过稠，板容易出现拖动或停顿造成的层纹；过稀，水蒸发后，板表面较粗糙。匀浆配比一般是硅胶G:水=1：2～3，硅胶 G:羧甲基纤维素钠水溶液=1:2。研磨匀浆的时间，根据经验来定，与空气湿度有关，一般通过拿起研棒时匀浆下滴的情况来判断，越稠越难下滴。匀浆的稀稠除影响板
层析技术(Layeranalise－technique)(1)
离子交换层析技术是以离子交换纤维素或以离子交换葡聚糖凝胶为固定相，以蛋白质等样品为移动相，分离和提纯蛋白质、核酸、酶、激素和多糖等的一项技术。 （一）原理 在纤维素与葡聚糖分子上结合有一定的离子基团，当结合阳离子基团时，可换出阴离子，则称为阴离子交换剂。如二乙氨乙基（Dicthy
层析技术(Layeranalise－technique)(3)
3．平衡 将DEAE―纤维素放入0.0lMol/L pH 7.4 PB液中（即起始缓冲液），静止1h，不时搅拌，待纤维素下沉后，倾去上清液或抽滤除去洗液，如此反复几次至倾出液体的pH值与加入的PB液的pH值相近时为止。4．装柱 层析柱的选择要大小、长度适当。一般而言，柱长和柱直径之比为10：1～20
层析技术(Layeranalise－technique)(2)
离子交换纤维素的优点为：①离子交换纤维素为开放性长链，具有较大的表面积，吸附容量最大；②离子基团少，排列稀疏，与蛋白质结合不太牢固，易于洗脱；③具有良好的稳定性，洗脱剂的选择范围广。 2．离子交换交联葡聚糖 离子交换交联葡聚糖也是广泛使用的离子交换剂，它与离子交换纤维素不同点是载体不同，常用交联葡聚
薄层色谱法(thin－layer－chromatography，TLC)
一、目的要求1. 了解薄层层析分离和鉴定有机物的原理和方法；2. 掌握薄层层析分离和鉴定有机物的操作步骤。二、基本原理薄层色谱具有设备简单、速度快、分离效果好、灵敏度高以及能使用腐蚀性显色剂等优点，是一种微量的分离分析方法。它可以与光谱或质谱结合起来，是一种很有发展前途的分析技术。薄层色谱是把吸附剂
ES－Cell－Culture－and－Manipulation
MediaHigh glucose DMEM (-pyruvate, -glutamine)20% Heat-inactivated Fetal calf serum (can vary by cell type, be sure!!)1X l-glutamine1X Penicillin/stre
Wright－Dust－Feeder－II－助力中国粉尘气溶胶研究
Wright Dust Feeder II 粉尘发生器助力中国粉尘气溶胶研究 自从Martin Wright研发Wright Dust Feeder II 粉尘发生器以来,它已经用于科学研究差不多60年的历史。 现在北京赛克玛环保仪器有限公司开始正式为中国 颗粒物研究人员提供Wright Du
Basics－of－Electrochemical－Impedance－Spectroscopy（四）
We will calculate two examples to illustrate a point about combining circuit elements. Suppose we have a 1 Ω and a 4 Ω resistor in series. The i