The murine cell line, 6606PDA and 7265PDA were a gift of Prof. Tuveson (University of Cambridge, UK) and were grown in DMEM high glucose medium (Biochrom GmbH, Berlin, Germany) as previously described [19 (link), 20 (link)]. The human MIA PaCa-2 cells were ordered from ATCC (LGC Standards GmbH, Wesel, Germany). The PSCs were isolated from the pancreas of C57BL/6J mice by collagenase digestion of the organ and by Nycodenz density gradient centrifugation as previously described [37 (link)]. These cells were expanded in Iscove's-medium (Biochrom GmbH, Berlin, Germany) supplemented with 17% fetal calf serum (FCS), 1% non-essential amino acids, 100 U/ml penicillin and 100 μg/ml streptomycin for one to two weeks. All experiments were performed with passaging the cells no more than 2 times.
Dmem high glucose medium
Manufactured by Harvard Bioscience
Sourced in United Kingdom
DMEM high glucose medium is a cell culture media formulation that provides a nutrient-rich environment for the growth and maintenance of various cell types. It contains a high concentration of glucose as the primary energy source, as well as essential amino acids, vitamins, and inorganic salts to support cell metabolism and proliferation.
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Lab products found in correlation
Iscove s medium, by Harvard Bioscience (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Mouse anti β actin antibody, by Merck Group (1 mentions)
Peroxidase linked anti mouse antibody, by Merck Group (1 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
2 protocols using dmem high glucose medium
1
Murine and Human Pancreatic Cell Lines
2
Western Blot Analysis of Apoptosis Markers
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The cells were seeded on 6 well plates in DMEM high glucose medium (Biochrom GmbH) supplemented with 10% fetal calf serum. On the following day the cells covered about 30% of the well surface and were incubated for another 24 or 48 hours with control medium or medium containing therapeutic agents (as described above). After separating the cell lysates on SDS polyacryl gels the proteins were transferred to a polyvinyldifluoride membrane (Immobilon-P; Millipore, Eschborn, Germany) as described previously 10 . The membranes were blocked with 2.5 % (wt/vol.) BSA and incubated overnight at 4 °C with rabbit-anti-cleaved caspase 3 (Cell Signaling, Danvers, MA, USA, code 9661, dilution: 1000x) or rb-anti PARP (Cell Signaling, Danvers, MA, USA, code 9542, dilution: 1000x) followed by incubation with a secondary peroxidase linked anti-rabbit (Cell Signaling, code 7074, dilution: 2000x for cleaved caspase 3 and 20000x for PARP Western Blot). For analysis of β-actin production, membranes were stripped, blocked by 2.5 % (wt/vol.) BSA and incubated with mouse anti-β-actin antibody (Sigma-Aldrich, code A5441, dilution: 20000x) followed by peroxidase-linked anti-mouse antibody (Sigma-Aldrich, USA; code A9044, dilution: 60000x).
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