Cellometer auto t4 plus cell counter

For amino acid consumption analysis, cell lines growing in log phase were trypsinized, counted and plated at a density of 100,000 cells/well of a six well dish and allowed to attach for 24 hr. The following day, cells were washed with 2 mL of PBS and then fed 2 mL of either RPMI or RPMI(10 µM cystine) media. 1 mL of media was immediately removed, spun for 3 min. at 845 g to remove any cells from the media, and then frozen at −80°C for later analysis. Cell number was also determined using a Cellometer Auto T4 Plus Cell Counter (Nexcelom Bioscience). 2 days later, media was harvested and cell number of the culture again determined. Amino acid concentration in the fresh or spent media was determined by GC-MS as described above in Media preparation and analysis. To calculate amino acid consumption rates, cell numbers at the initial and day two time points were used to fit an exponential growth function, and integration of these curves yielded the number of (cell · days) by which the media was conditioned. Changes in amino acid concentration for each culture were normalized to the integrated growth curve of each culture to yield amino acid consumption/release per cell per unit time.

An in vitro test was performed to examine the cell cytotoxicity of the co-incubated PBA-based contact lens. ARPE-19 cells (a human retinal pigment epithelial cell line) were routinely cultured and passaged in a cell culture dish at 37 °C and 5% CO₂ using a DMEM medium supplemented with 10% Ham’s F12 medium (Gibco, Thermo Fisher). The cells were harvested after 24 h and then incubated with individual glucose contact lenses for an additional 8 h. After being co-incubated for 8 h, the cells were stained with trypan blue and quantified as percentages of viable cells with the Cellometer^® Auto T4 Plus cell counter (Nexcelom Bioscience, Lawrence, MA, USA).

Measurement of intracellular DCFDA fluorescence was performed using the Abcam DCFDA Cellular Reactive Oxygen Species Detection Assay Kit (Abcam, Cambridge, MA, ab113851) according to the manufacturer’s instructions. Briefly, 25,000 A549 cells were plated in each well of a 96 well plate in 200 µL RPMI without phenol red (Sigma Aldrich). Cells were allowed to attach for 8 hr. Subsequently, the media on the cells was changed to 200 µL fresh media containing DMSO, CB-839 and NAC as indicated. After 24 hr of treatment, the cells were washed twice with 200 µL of 1x Buffer included in the assay kit. Cells were then incubated with 100 µL of 1x Buffer containing 10 µM DCFDA for 45 min. at 37°C. An unstained control was included, and incubated with 100 µL of 1x Buffer alone. After staining, the DCFDA containing solution was removed and cells were resuspended 100 µL of PBS. DCFDA fluorescence was measured using an Infinite M200Pro plate reader (Tecan) in fluorescence mode with excitation at 485 and emission at 535. Cells were then detached with trypsin-EDTA solution and counted using a Cellometer Auto T4 Plus Cell Counter (Nexcelom Bioscience). The ratio of DCFDA signal intensity to cell number was subsequently computed.

Cellular proliferation rate in different media and drug conditions was determined as previously described (Sullivan et al., 2015 (link)). Briefly, cell lines proliferating in log phase in RPMI medium were trypsinized, counted and plated into six well dishes (Corning Life Sciences) in 2 mL of RPMI medium and incubated overnight. Initial seeding density was 20,000 cells/well for A549 cells, or 50,000 cells for MCF7, AU565 and MDA-MB-468 cells. The next day, a six well plate of cells was trypsinized and counted to provide a number of cells at the start of the experiment. Cells were then washed twice with 2 mL of phosphate buffered saline (PBS), and 8 mL of the indicated media premixed with indicated compounds or vehicles was added. This large volume of media was chosen to prevent severe nutrient depletion, especially when adding adult bovine serum medium. Cells were then trypsinized and counted 4 days after adding the indicated medias. Proliferation rate was determined using the following formula: Proliferation rate in doublings/day = [Log2(Final Day 4 cell count/Initial Day 0 cell count)]/4 days. Cells were counted using a Cellometer Auto T4 Plus Cell Counter (Nexcelom Bioscience, Lawrence, MA).