By quantifying the fluorescence intensity of doxorubicin (DOX), the intracellular accumulation of doxorubicin (DOX) was determined. At a density of 5 × 105 cells per well, K562 or K562/ADR cells were seeded in 24-well plates. The cells were then pre-treated with either 12.5 or 25 µg/mL of PGG or 10 or 20 µM of verapamil (VP) and cultured at 37 °C with 5% CO2 for a period of 24 h. Subsequently, 10 µM of DOX was added to each well, and the cells were allowed to incubate for an additional 1 h at 37 °C. Following the incubation with DOX, the cells were harvested, washed twice with ice-cold PBS, and lysed with a lysis buffer (CelLytic™ M Cell Lysis, Sigma-Aldrich, St. Louis, MO, USA). The intracellular fluorescence intensity associated with DOX was finally measured using a multi-mode microplate reader (Molecular Devices SpectraMax® i3x, San Jose, CA, USA). The fluorescence intensity was measured at excitation and emission wavelengths of 480 and 590 nm, respectively.
Cellytic m cell lysis
Manufactured by Merck Group
Sourced in United States, Australia
CelLytic™ M Cell Lysis is a reagent designed for the gentle and efficient lysis of mammalian cells. It is a non-ionic detergent-based solution that effectively solubilizes cellular proteins while preserving their natural structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax i3x, by Molecular Devices (1 mentions)
Trypan blue solution, by Thermo Fisher Scientific (1 mentions)
Snapwell cell culture inserts, by Corning (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Calu 3 cells, by American Type Culture Collection (1 mentions)
2 protocols using cellytic m cell lysis
1
Quantifying Intracellular Doxorubicin Accumulation
2
Calu-3 Cell Culture Protocol
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Calu-3 cells were purchased from the American Type Cell Culture Collection (ATCC, Rockville, US). Ciprofloxacin HCl was used as supplied (MP; Biomedical Australasia Pty Limited, NSW, Sydney, Australia). Non-essential amino acids solution, CelLytic™ M Cell Lysis, protease inhibitor cocktail and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Sydney, Australia). Other cell culture reagents including trypsin-EDTA solution (2.5g/L trypsin, 0.5g/L EDTA), Dulbecco's Modified Eagle's Medium (DMEM, without phenol red and L-glutamine, including sodium bicarbonate and 15mM HEPES), Trypan blue solution (0.4% w/v), Phosphate buffered saline (PBS), L-glutamine solution (200mM), fetal bovine serum (FBS), Hank's balanced salt solution (HBSS) were obtained from Invitrogen (Australia). Snapwell cell culture inserts (1.12 cm 2 polyester, 0.4µm pore size) were purchased from Corning Costar (Lowell, MA, USA) and all other sterile culture plastic wares were from Sarstedt (Adelaide, Australia). All solvents used were of analytical grade and were supplied by Biolab (Victoria, Australia).
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