When monolayers had matured and reached a RTE plateau, Ussing chamber recording

From day 26 to day 35 the DNR containing med ium was changed once per week. At day 35 there were very few remaining JAK3 阻害剤 viable cells, so cells were separated over Histopaque to exclude dead cells and cell debris. The remaining cells were re suspended in 10 ml of medium containing 10 nM DNR up until day 45 when cell growth was seen. Cells were maintained at this drug concentration until their growth rate approached that of untreated OCI AML3 cells. Over the next 10 weeks cells were exposed to gradually increasing concentra tions of DNR up to a final concentration of 15 nM. By using the R123 accumulation assay these cells were con firmed to have increased levels of Pgp function and were subsequently cloned by limiting dilution. Cells were serially diluted in 96 well plates down to a concen tration of 1 cell/well in medium containing 15 nM DNR.<br><br> Any cells showing outgrowth were then gradually supplier LDE225 cultured in medium containing 15 nM DNR up to suffi cient numbers to be able to assay for Pgp protein and function. One clone showed an increase in both Pgp protein expression and function. This clone was named OCI AML3DNR and was then cultured under standard conditions with 15 nM DNR and also cryo preserved for future use. DNR was removed from culture medium 7 days prior to any experimental procedure. OCI AML3/OCI AML3DNR/OCI AML6. 2 genetic analysis DNA was prepared using a QIAamp DNA blood mini kit and 5 ng DNA was amplified using the Powerplex 16 System to assess STR. The products were run on a 3130 Genetic Analyser and the data analysed using GeneMap per ID v3. 2 software.<br><br> Patient LY2157299 TGF-beta 阻害剤 samples Presentation blood or bone marrow samples from multi ple centres were obtained at diagnosis from patients with AML and taken into pre servative free heparin or into EDTA tubes. All samples were pre treatment and only samples received within 48 hours of being removed from the patient were analyzed. Use of these samples was approved by the Nottingham 1 Research Ethics Committee. Mononuclear cells were isolated using a standard density gradient/centrifugation method with Histopaque and cryopreserved in liquid nitrogen. For analysis, cryopreserved samples were thawed and rested in culture medium enriched to 20% FCS for 90 minutes before experimental procedures. Thawed and rested samples were then subjected to viability analysis using trypan blue exclusion and only samples with 85% post rest viability were used.<br><br> Pgp protein expression MRK 16 mAb For Pgp protein expression cells were harvested, washed in PBSAA, and 2 105 cells incubated with 1 ug MRK 16 anti Pgp monoclonal antibody or IgG2a isotype control for 30 min at RT. Cells were washed x3 in PBSAA and blocked in 80 ul 20% normal rabbit serum for 30 min on ice. 5 ul FITC conjugated goat anti mouse secondary antibody was added and cells incubated for 30 min on ice. Cells were washed twice in PBSAA and cell line data collected using a FACS cali bur. Patient samples had the added step of the addition of 5 ul CD45PerCP and 5 ul Normal Mouse serum before being washed twice in PBSAA and collected using a FACS calibur. Labelling the patient sam ples with CD45PerCP allowed the leukemic cells to be gated.