公开(公告)号：EP2322913A3

公开(公告)日：2012-10-03

申请号：EP10184182.3

申请日：2004-09-08

申请人： Seahorse Bioscience, Inc.

发明人： Teich, Jay S ,  Neilson, Andy C ,  Sweeney, Michael R ,  Uhl, Geoff

IPC分类号： G01N21/64 ,  G01N21/03 ,  B01L3/00 ,  C12M1/34 ,  G01N21/85

CPC分类号： B01L3/5085 ,  B01L2300/046 ,  B01L2300/0654 ,  B01L2300/0663 ,  B01L2300/0829 ,  B01L2300/0851 ,  B01L2300/12 ,  C12M23/12 ,  C12M23/22 ,  G01N21/0303 ,  G01N33/5023

摘要： An apparatus for analysis of cells disposed in media in multiple wells of a multi-well plate, the comprises: a plurality of barriers for insertion into respective wells of the multi-well plate, each barrier comprising a barrier surface that creates, when inserted into the respective well, a sample chamber having a reduced volume of medium less than 50% of the original volume of medium in the wells, and disposed on barrier surfaces, fluorescent sensors for analyzing a constituent of the medium disposed about the cells in the respective sample chamber.

公开(公告)号：EP2322913A2

公开(公告)日：2011-05-18

申请号：EP10184182.3

申请日：2004-09-08

申请人： Seahorse Bioscience, Inc.

发明人： Teich, Jay S ,  Neilson, Andy C ,  Sweeney, Michael R ,  Uhl, Geoff

IPC分类号： G01N21/64 ,  G01N21/03 ,  B01L3/00 ,  C12M1/34 ,  G01N21/85

CPC分类号： B01L3/5085 ,  B01L2300/046 ,  B01L2300/0654 ,  B01L2300/0663 ,  B01L2300/0829 ,  B01L2300/0851 ,  B01L2300/12 ,  C12M23/12 ,  C12M23/22 ,  G01N21/0303 ,  G01N33/5023

摘要： Table 5 demonstrates that, as expected, cell proliferation results in an increase in oxygen consumption and the rate of extracellular acidification.
Example 6
Measurement of G-Protein Coupled Receptor Activation in CHO-M3 Cells:
Previous studies have shown that stimulation of transmembrane receptors often causes a rapid increase in extracellular acidification rate, resulting primarily from acute activation of ion exchange pumps. In this experiment, the prototype device was used to detect a change in extracellular acidification rate following treatment of cells with a receptor agonist.
Chinese hamster ovary (CHO) cells were transfected to over-express the muscarinic receptor subtype m3. The prototype device described in Example 1 was then used to monitor O 2 consumption, CO 2 production, and extracellular acidification rates, following treatment with the well-known, general acetylcholine receptor agonist, Carbachol.
Materials and Methods: Cell culture reagents were obtained from Gibco BRL (Grand Island, NY). Carbachol was purchased from Sigma Chemical Co. (St. Louis, MO). Biearbonate-free DMEM medium was obtained from Specialty Media (Phillipsburg, NJ). Polycarbonate membrane snapwells (12 mm diameter, 3 µm pore size) were obtained from Corning (Corning, NY). CHO cells expressing m3-muscarinic receptors (CHO-M3 cells) were obtained from the American Type Tissue Culture (ATCC; Manassas, VA). Cells were cultured in Ham's F-12 medium supplemented with 10% fetal bovine serum (Hyclone), 1% GlutaMax and 0.1 % Gentamicin and were maintained in a 5% CO 2 incubator. Cells were subcultured when they reached 80% confluency. CHO-M3 cells were seeded at a density of 2x10 5 onto a snapwell 24 hours prior to use. Immediately prior to testing, cells on snapwells were switched to bicarbonate-free DMEM medium combined with 3.7 g/l NaCl to maintain osmolarity (medium pH 7.4 - 7.5).
Protocol Description: Probes were calibrated immediately prior to testing. The bottom of the test vessel was filled with bicarbonate-tree medium. The snapwell was removed from a 5% CO2 incubator, and the regular growth medium (Ham's F-12) was replaced with bicarbonate-free DMEM medium. Thereafter, the snapwell was placed into the test vessel. Bicarbonate-free medium was pipetted onto the top of the snapwell, and the cover piece of the test vessel was placed gently on top of the snapwell and screwed into place, compressing the assembly. The probe software was started, and the pH, CO 2 and O 2 analytes were measured over

摘要翻译： 用于在多孔板的多个孔中的媒体处理完毕细胞的分析的装置，所述步骤包括：用于插入屏障的多元到多孔板的respectivement孔，每个阻挡包括阻挡表面并创建，当被插入到 的respectivement井，其具有在孔中培养基的初始体积的介质小于50％的减小的体积的样品室，以及设置在屏障表面，荧光传感器，用于分析设置在所述respectivement样品中的细胞的培养基的组成 室。