Synergy ht microplate spectrophotometer

The PKIS1, PKIS2, and Selleck libraries were obtained via the University of Wisconsin Carbone Cancer Center’s Small Molecule Screening Facility. Overnight cultures were back-diluted 1:100 into fresh TSB medium containing 10 μg/mL ceftriaxone and either library compounds (final concentration: 20 μM in 2% DMSO) or DMSO (final concentration: 2%). Growth was measured as an optical density at 600 nm (OD₆₀₀) on 15 min intervals for 12 h in a 96-well format using an Eon microplate spectrophotometer or Synergy HT microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT) (growth conditions: 37 °C, linear shaking). Each compound was screened twice. Percent inhibition was calculated as (1 – (OD_X/OD_CRO)) × 100, where OD_X is the end point OD₆₀₀ for a culture treated with both ceftriaxone and compound X, and OD_CRO is the end point OD₆₀₀ for a culture treated with ceftriaxone alone. Compounds that inhibited growth three standard deviations greater than the library mean were further verified for dose responsiveness and β-lactam dependence.

To test the cytotoxic effects, the 1 ml of APCS-gel, fibrin gel or Tisseel was immersed in 10 ml corneal epithelial medium or corneal stromal cell medium. After 24 hours at 37 °C in the tissue culture incubator, APCS-gel, fibrin gel and Tisseel were removed respectively, and the remaining media were stored at 4 °C. For the cytotoxicity assay, primary RCECs (1×10³) or RCSCs (1×10³) were seeded into 96-well plates and cultured with these media that were incubated with hydrogels, fibrin or Tisseel [24 (link), 32 (link)]. Each experiment was done in triplicate. After 1, 3, 5, and 7 days, the proliferation activity of the cells was quantitatively determined by MTT assay. The optical density (OD) of absorbance at 490 nm was measured by a microplate reader (Synergy HT, Microplate Spectrophotometer, Biotek, USA).