Dose Escalation: A Method for Developing Drug Resistance in Cancer Cells

Samenvatting

Source: Yamaoka T. et al., Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib. J. Vis. Exp., 2019

The video describes the development of drug resistance in cancer cells by exposing cells to escalating doses of a drug after calculating IC₅₀ values of the drug previously with MTT assay. The sample protocol describes the step-by-step process of developing drug-resistant cells.

Protocol

1. Establishment of Three Independent Afatinib-resistant PC-9 Cell Lines

1. Determination of the initial afatinib exposure concentration for PC-9 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay.
   1. Culture PC-9 cells in growth medium containing fetal bovine serum (10%), penicillin (100 U/mL), and streptomycin (100 µg/mL) in a cell-culture treated 10-cm dish in a 5% CO₂ incubator at 37 °C.
   2. Resuspend PC-9 cells at 4 x 10⁴ cells/mL in growth medium and then seed at 50 µL/well in a 96-well microplate. The final concentrati....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
FBS	gibco	26140-079
Pen Strep	gibco	15140-163
cell-culture treated 10 cm dish	Violamo	2-8590-03
afatinib	Selleck	S1011
96 well microplate	Thermo	130188
CellTiter 96	Promega	G4100	Non-Radioactive Cell Proliferation Assay; Dye solution and Solubilization/Stop solution
NanoDrop Lite spectrophotometer	Thermo	NA	spectrophotometer
GraphPad Prism v.7 software	GraphPad, Inc.	NA	a statistical software
DMSO	Wako	043-07216