High glucose dulbecco s modified eagle medium dmem

HPLC-grade solvents were purchased from Merck (Steinheim, Germany) and Fisher Scientific (Waltham, MA, USA). Ultrapure water (18.3 MΩcm), distilled, and deionized water (Milli-Q water) were purified using a Milli-Q system (Merck-Millipore, Bedford, USA). High glucose Dulbecco’s modified Eagle Medium (DMEM) was purchased from Sigma (St. Louis, MO, USA). Trypsin-EDTA, L-glutamine, penicillin–streptomycin solution, and heat inactivated fetal bovine serum (FBS) were obtained from Biochrom KG, (Berlin, Germany). Human Caucasian breast adenocarcinoma cell line (MCF-7) was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured as monolayers in complete medium Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, Steinheim, Germany) at 37 °C in an atmosphere of 5% (v/v) CO₂ and 95% at relative humidity. Cells were seeded in 75 cm² plastic tissue culture flasks and cultured in DMEM supplemented with 10% FBS, 2 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, washed with phosphate buffered saline (PBS) and harvested by trypsinization with 0.05% (w/v) trypsin in PBS containing 0.02% (w/v) EDTA (Sigma, St. Louis, MO, USA).

The trapped single cells were cultured in situ for multiple generations with time-lapse imaging to analyze the growth dynamics. The microfluidic chip with 30 μm deep filtration channels, either with or without a pneumatic valve, was fixed in a stage-top incubator (Tokai Hit STX, Fujinomiya, Japan) on the motorized microscope. The incubator was used to precisely regulate the temperature (37 ± 0.1 °C), concentration of CO₂ (5%), and moisture (95%).
After a positive pressure of 2 bar was applied to the gas channel to deform the PDMS membrane, K562 cells were injected and captured by the filtration array using a similar protocol to that mentioned above (Figure 1B Capturing). After the complete rinsing of the chip using media, the membrane was deactivated to the normal state (B rinsing). Then, high-glucose Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich, Missouri, USA) with 10% fetal bovine serum (FBS) (JRH Biosciences, Kansas, USA) and 1% penicillin-streptomycin (P-S) (Sigma-Aldrich, Missouri, USA) stored in a 5 mL syringe was continuously injected into the chip at 0.3 μL/min through the media inlet (B culturing) for up to 7 days. Meanwhile, approximately 30 positions with distinct trapped single cells were imaged every 3 min using phase-contrast mode with a 10X objective. The media in the syringe was protected from light at room temperature during the whole culture period.

High glucose Dulbecco’s modified Eagle Medium (DMEM) was purchased from Sigma (Steinheim, Germany). Trypsin-EDTA, L-glutamine, penicillin–streptomycin solution and heat inactivated fetal bovine serum (FBS) were obtained from Biochrom KG (Berlin, Germany). U87MG brain glioblastoma was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured as monolayers at 37 °C in a humidified atmosphere of 5% (v/v) CO₂ and 95% relative humidity. Cells were seeded in 75-cm² plastic tissue culture flasks and cultured in DMEM supplemented with 10% FBS, washed with phosphate buffered saline (PBS) and were harvested by trypsinization with 0.05% (w/v) trypsin in PBS containing 0.02% (w/v) EDTA. SCID mice were xenografted at two weeks of age with U87MG cells subcutaneously at the right side of the thorax. Tumors were inoculated after injection of 6 × 10⁶ U87MG cells in SCID mice, which were previously grown in DMEM.