Celltracker green cmfda dye

Silicon wafers (p-type, single side polished, Ø =100 mm) were purchased from Silicon Materials. Positive tone photoresist and developer were purchased from AZ Electronic Materials. Piranha solution (H₂O₂:H₂SO₄, 1:3) was produced by mixing 1 part of sulfuric acid, from Sigma-Aldrich, with 3 parts of hydrogen peroxide, from Honeywell. SU-8 photoresist and developer were purchased from Microchem and polydimethylsiloxane (PDMS) Sylgard 184 elastomer kit from Dow Corning. Phosphate buffered saline (PBS), Minimum Essential Medium a (a-MEM), sodium pyruvate and 0.25% trypsin in ethylene-diamine-tetraacetic acid (EDTA) were obtained from Gibco, Life Technologies. Fetal bovine serum (FBS) was purchased from Lonza and CellTracker™ Green CMFDA dye from Molecular Probes, Life Technologies. N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid (HEPES), Cytochalasin D and CF™ 568 maleimide were purchased from Sigma-Aldrich. Human osteosarcoma cell line MG-63 (ATCC® CRL1427™) was obtained from ATCC-LGC.

The vessels-on-a chip were made using the devices developed by the lab of Beebe, as previously described (8 (link)). Minor alterations to the protocol were made. Briefly, the devices were coated with 1% PEI (Polysciences Inc., Warrington, PA, USA, #23966) and incubated for 10 minutes at room temperature (RT). Sequentially chambers were coated with 0.1% glutaraldehyde (Merck, Darmstadt, Germany, #104239), washed 5x with water for injection (WFI; Gibco, #A12873-01) and air-dried. Collagen-1 was prepared according to the protocol above. 10 µl Collagen-1 was added to each chamber and polymerized for 30 minutes at 37°C, 5% CO₂. PBS-drenched cotton balls were added to the device to control humidity of the device. Rods were removed using tweezers and EGM-2 was added to the lumen. HUVECs were washed twice with PBS, trypsinized for 5 minutes, treated with trypsin neutralizing solution (TNS; Lonza, cat CC-5002) and centrifuged for 5 minutes at 200xg. HUVECs were then stained using CellTracker™ Green CMFDA Dye (1 µM; Molecular Probes, cat C7025) according to manufactures protocol, washed twice with PBS and pelleted. HUVECs were then resuspended to a concentration of 15*10⁶ cells/mL. 5µl of cell suspension was added to each lumen and placed in head-over-head at 37°C, 5% CO₂ for 2 hours at 1 RPM. Vessels were matured for 2 days with medium replacement twice daily.

Two days after seeding, HMEC-1 cells have formed the confluent monolayers in the wells of 24-well plates and were subjected to a suitable treatment. Mammary epithelial cells (MECs) were starved overnight and on the assay day were labeled with CellTracker Green CMFDA Dye (Molecular Probes). Just before the assay, the HMEC-1 cells culture medium was substituted with the adhesion medium (MCDB 131 containing 1 mM CaCl₂, 1 mM MgCl₂, and 1% bovine serum albumin). Subsequently the aliquots of MECs (1 × 10⁵/well) were applied onto the endothelial monolayers and allowed to adhere for 1 h in a humidified incubator (37 °C, 5% CO₂). Non-adherent cells were washed away by PBS. The adherent cells were lysed by 2% SDS, and the fluorescence was measured using the Wallac 1420 VICTOR2 Multilabel Counter at 492 nm of excitation and 517 nm of emission wavelengths.

To form three dimensional (3D) cell aggregates, cells, with or without miRNA/siRNA transfection, were incubated overnight in RPMI with 10% FBS, with a magnetic gold–polymer–iron oxide hydrogel (Nanoshuttle-PL, NS) (n3D Biosciences, Houston TX) to allow the attachment of NS to the cells [36 (link), 37 (link), 74 (link)]. 5 uM of CellTracker Green CMFDA Dye (Molecular Probes) was added to the cells and incubated for 30 minutes. The cells were washed with PBS, trypsinized, resuspended in RPMI with 10% FBS, and then magnetically levitated to the air-water interface for 4 hours, where they formed 3D structures with an ECM. Next, the 3D structures were mechanically disrupted by pipetting, and the resulting suspensions of aggregates were transferred to a 96-well plate, where they were formed or ‘printed’ into either a dot or a ring shape by placing a dot or ring magnet ‘drive’ underneath the plate for 15 minutes. Using either phase contrast or fluorescence, the 3D culture area was automatically imaged over time by an IncuCyte ZOOM live-cell imaging system (Essen BioSciences, Ann Arbor, Michigan), and the area of the dense 3D spheroidal regions was recorded for each well.

NHOKs (2 × 10⁵/well) were treated with different doses of H₂O₂ for 1 h or 8 h and then harvested. The cells were incubated at 37 °C for 20 min with 500 μL of 1X 20-μM of CellTracker Green CMFDA Dye (Molecular Probes, Eugene, OR, USA) and then centrifuged at 1200 rpm (241 g) for another 5 min. They were then washed three times with 500 μL of PBS, resuspended, and transferred to flow tubes. The glutathione (GSH) content, determined using CMFDA (green, Ex = 522 nm; Em = 595 nm), was quantitated using a flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) at the FL1-H channel, as previously described [22 (link)].