Quadruplicates of 1104 EGFP SKBR3 selleck compound cells were seeded in black walled 96 well plates with increasing numbers of AT MSCs and cultured for 6 days. Green fluorescence was directly pro portional to the number of viable tumor cells within the wells and the fluorescence value in the untreated cells was set to 100% by default. Experiments were evaluated as mean of quadruplicates SD. In order to dissect the role of SDF 1 CXCR4 axis in proliferation of EGFP SKBR3 cells in cocultures with AT MSCs, specific inhibitor of this signaling axis AMD 3100 was used. Final concentra tion of 5 ug ml AMD 3100 was added to EGFP SKBR3 cells alone, cultured in MSC CM or in coculture with AT MSCs. The effect on proliferation was evaluated as a relative fluorescence as described above.
Relative cell viability was evaluated by CellTiter Glo Luminescent Cell Viability Assay based on the ATP quantitation representa tive of metabolically active cells. Quadruplicates of Inhibitors,Modulators,Libraries 6103 SKBR3 cells per well were seeded in 96 well plates over night. Diluted MSCs CM was added to the adherent tumor cells on the next day. Relative proliferation was determined Inhibitors,Modulators,Libraries on LUMIstar GALAXY reader. Values were expressed as mean rela tive luminescence SD, when luminescence of control cells was taken as reference. Experiments were repeated at least twice with similar results and a representative result is shown. Chemosensitivity Following drugs were used, 5 fluorouracil, doxorubicin and cis platin. For the evalu ation of chemosensitivity, either 6103 EGFP SKBR3 cells alone or mixed with AT MSCs were seeded in 96 well plates.
On day 0, treatments were started with doxorubicin, 5FU or cis platin. The chemosensitivity was determined Inhibitors,Modulators,Libraries by fluorescence measurements as described above 6 days later. Experiments were evaluated as means of three different experiments run in quadruplicates and the relative fluorescence in untreated cells was taken as 100% by default. Alternatively, 8103 EGFP SKBR3 were seeded in 96 well plates overnight and treated with the drugs diluted in MSCs CM. Relative fluorescence and cell proliferation was determined as above. Caspase 3 7 assay Quadruplicates of 2104 SKBR3 per well were seeded in 96 well white walled plates overnight. Doxorubicin or 5FU diluted in MSC CM or culture media was added to the cells for the indicated period of time and a Caspase 3 7 activity was determined Inhibitors,Modulators,Libraries by the Caspase Glo 3 7 Inhibitors,Modulators,Libraries Assay on LUMIstar GALAXY reader at indicated timepoints.
Values were Volasertib FDA determined as mean values of RLU SD. Annexin V assay In order to quantify a proportion of viable, apoptotic and necrotic cells in cocultures, adherent AT MSCs were labeled with 5 uM carboxy fluorescein diacetate, succinimidyl ester in a serum free DMEM for 15 min at 37 C. Medium was replaced for standard culture medium to incubate overnight.
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