Experiment Id: 65

Pattern Id: MT215

Clone number:

GenBank Acc:

Rationale: Drug resistance is a major reason for the failure of cancer chemotherapy. One widely studied mechanism of drug resistance involves the ability of P-glycoprotein to act as a drug efflux pump.

Investigator(s):

Fojo, Dr. Tito

National Cancer Institute

Method: Rhodamine efflux

Unit: Difference in rhodamine flourescence intensity (a difference of 256 channel numbers represents a 10-fold change in fluorescence intensity).

Protocol: Suspensions of logarithmic phase cells were obtained from tissue culture plates by trypsinization. During the accumulation period, 4 aliquots of cells were resuspended in rhodamine-containing medium (Improved minimum essential medium with 10% fetal calf serum and 0.5 ug/ml rhodamine 123) and incubated with or without 3.0 ug/ml cyclosporin A at 37o in 5% CO2 for 30 min. After the accumulation period, efflux was initiated by sedimentation at 600 xg and resuspension in rhodamine-free medium (Improved minimum essential medium with 10% fetal calf serum ), with or without 3.0 ug/ml cyclosporin A. The efflux was carried out at 37o in 5% CO2 for 90 min. At the end of both the accumulation and efflux periods, cells were sedimented, washed in ice-cold Hank's buffered salt solution, placed in Hank's buffered salt solution with 10% fetal calf serum on ice, and kept in the dark until flow cytometric analysis. Samples were analysed on a FACScan flow cytometer equipped with a 488-nm argon laser. The green fluorescence of rhodamine 123 was collected after a 530-nm band pass filter. Samples were gated on forward scatter versus side scatter to exclude cell debris and clumps. A minimum of 6000 events were collected for each sample. Histograms were generated for each cell line and the median rhodamine fluorescence obtained for each peak were identified by channel number, and this number (denoting the logarithm of fluorescence intensity) was used to indicate the amount of rhodamine in the cells in each incubation condition. A difference of 256 channel numbers represents a 10-fold change in fluorescence intensity.

Target Data:

Compare:

Genecard

A database of human genes, their products and their involvement in diseases. It offers concise information about the functions of human genes.

Unigene

An experimental system for automatically partitioning GenBank sequences into a non-redundant set of gene-oriented clusters.

Entrez Gene

Presents information on official nomenclature, aliases, sequence accession numbers, phenotypes, EC numbers, MIM numbers, UniGene clusters, map information, Gene Ontology annotations, and relevant web sites. Replaces LocusLink.

OMIM

A catalog of human genes and genetic disorders.

PubMed

Search PubMed for articles on:

CGAP

CGAP provides information on gene expression, SNPs, functional pathways and more in normal, precancerous and cancer cells.