点赞

收藏

分享

In vitro Assessment of Transporter-Mediated Uptake in Plated Cryopreserved Hepatocytes

互联网2013-11-13

523

实验设备

37°C water bath

37°C / 5% CO₂ humidified incubator

Orbital shaker placed inside incubator

实验步骤

1. Prepare compound stocks: test articles (TA) and positive control(s) (PC) dissolved in an organic solvent such as methanol or DMSO to desired concentration, such as 1 mM.

2. In separate conical tubes, add the test compounds and positive control(s) to warm Incubation Medium to yield the desired working concentration(s). For example, prepare 1 μM by adding 20 μL of 1 mM test article stock solution to 20 mL of Incubation Medium. Note: if DMSO is used as a solvent, the concentration should not exceed 0.1%, with a maximum of 1% in the final Incubation Medium.

3. Remove the wash medium from the hepatocytes and replace with an appropriate volume of Incubation Medium containing the TA or PC for the appropriate time point. Incubate on an orbital shaker for 0, 1, 2, 4, 6 and 8 hr at 80-120 rpm for a 24-well plate. Some low-turnover compounds typically require up to 24 hr incubation periods.

4. Stop the incubations by addition of the appropriate quenching solvent. Mix briefly and collect samples and eithe freeze at -70°C, or directly extract as per analytical methods. If plate is to be returned to incubator, aspirate remaining quenching solvent.

5. Analyze parent disappearance (and metabolite formation as desired) by analytical method(s) of choice, typically HPLC or LC-MS/MS.

6. Determine the in vitro half-life (t_1/2 ) of the parent compound by regression analysis of the percent parent disappearance vs. time curve.

7. Intrinsic clearance in vitro (Cl_{int in vitro} ) can be calculated according to the equation: Cl_{int in vitro} = kV/N, where k = 0.693/t1/2, V = incubation volume and N = number of hepatocytes per well (Table 1).

8. Clint in vitro may be scaled to in vivo predictions according to Obach and McGinnity.

Table 1. Parameters used in intrinsic clearance in vitro (Clint,in vitro) calculations for human hepatocytes. The assumption for N is that all hepatocytes adhered to the well with respect to seeding density.

Plate format	Seeding density (10⁶ total cells/mL)	Incubation volume V (mL)	Hepatocytes per well N (10⁶ cells)
12-well	Either 0.7 / 0.8 / 0.9	1.0	0.7 / 0.8 / 0.9
24-well	Either 0.7 / 0.8 / 0.9	0.5	0.35 / 0.4 / 0.45
48-well	Either 0.7 / 0.8 / 0.9	0.2	0.14 / 0.16 / 0.18
96-well	Either 0.7 / 0.8 / 0.9	0.1	0.07 / 0.08 / 0.09

注意事项

Review this protocol, as well as the protocol, Thawing and Use of Plateable and Suspension Cryopreserved Hepatocytes, to ensure you have all the necessary reagents and equipment prior to starting the procedure. Once thawed, cryopreserved hepatocytes must be used immediately and will not maintain viability if refrozen.

Use universal safety precautions and appropriate biosafety cabinet when handing primary hepatocytes.

ad image

ad image

相关产品推荐

SLC31A1/SLC31A1蛋白/Copper transporter 1 Short name: hCTR1 Solute carrier family 31 member 1蛋白/Recombinant Human High affinity copper uptake protein 1 (SLC31A1) (Active)重组蛋白

￥69

Gel-Based Customizable In Vitro E3 Ligase Activity Kit (VU1-HRP)

￥44

EZElisa™人透明质酸介导的运动受体（HMMR）ELISA试剂盒-EZElisa™ Human Hyaluronan Mediated Motility Receptor (HMMR) ELISA Kit

￥6448

B0182905珀金埃尔默不锈钢制垫片Gold Plated Copper Seals, Pkg. 10 PerkinElmer

￥1770

fur/fur蛋白Recombinant Escherichia coli Ferric uptake regulation protein (fur)重组蛋白Short name: Ferric uptake regulator蛋白

￥2328

相关问答

问

求文献In vitro and ex vivo models of adipocytes

问

in gel kinase assay和in vitro kinase assay的区别

问

in vitro kinase assay和In Gel Kinase Assay的区别

推荐阅读

Assessment of CYP3A4 Time-Dependent Inhibition in Plated and Suspended Human Hepatocytes

Heparan Sulfate Proteoglycan-Mediated Polyamine Uptake

In Vitro Cytotoxicity Assessment

关于丁香通

公司信息

个人用户

企业机构

无忧采购轻松科研

无忧采购轻松科研

提问

扫一扫

丁香实验小程序二维码

实验小助手

丁香实验公众号二维码

扫码领资料

反馈

TOP

打开小程序