Alamarblue

For alamarBlue (Life Technologies, Grand Island, NY, USA) dye-based cell growth assays, cells were seeded at 5×10³ cells/100 µl media (NALM6 and RCH-ACV cells) or at 2×10³ cells/100 µl media (REH cells) in triplicates in 96-well plates at 3 days after transduction. At 7 days after transduction, 10 µl alamarBlue was added to each well and plates incubated (37°C, 4 h) before reading using a VictorX3 (PerkinElmer, Waltham, MA, USA; 530/580 nm excitation/emission filters). For trypan blue exclusion cell counts, 2.5×10⁵ cells/ml were seeded in each well of a 96-well plate day on day 3 after transduction. 10 µl of cell suspensions were removed at each time point and counted using a hemocytometer.

Cells seeded into a 96-well plate were treated with JTE-607 at various conc. Three replicates were used for each cell type. Control wells were treated with DMSO. After three days of treatment, AlamarBlue reagent (Thermo Fisher Scientific, Cat #: DAL1100) was added into each well at the final conc. of 10% (v/v). Cells were incubated for 2–4 h at 37 °C. AlamarBlue fluorescence intensity was detected by a microplate reader (Perkin Elmer, 2104 EnVision) with the excitation and emission wavelengths of 540 nm and 595 nm, respectively. For U937/iFIP1 single clones, cells were first induced with 2 μg/mL Dox for two days before JTE-607 treatment.

For quantitative analysis of drug cytotoxicity, triplicate wells containing Hep3B cells (1 × 10⁴ cells/well) that had been pretransduced with AMLV-CD and/or GALV-CD at an MOI of 0.01 and maintained for 10 days were cultured in 96-well tissue-culture plates with various concentrations of 5FC (Wako Pure Chemical Industries, Osaka, Japan) and ganciclovir (GCV; Wako Pure Chemical Industries). On day 3, the viable cell numbers in the triplicate cultures were measured using Alamar Blue (Alamar Biosciences Inc., Sacramento, CA, USA) according to the manufacturer’s instructions. Briefly, 40 μL of Alamar Blue was aseptically added to the cultures, and the plates were returned to the incubator for 3 h. Fluorescence was measured by an ARVO X4 multilabel plate reader (PerkinElmer Japan, Tokyo, Japan) with excitation wavelength of 544 nm and emission wavelength of 590 nm. The percentage of viable cells was determined by calculation of viable cell fluorescence in wells containing 5FC and GCV relative to that in wells without 5FC and GCV. Combinatorial effects were assessed by combination index (CI) values using CalcuSyn software (Biosoft, Cambridge, UK) in different fraction-affected points, to show the relative levels of suppressed cell viability. Values of CI < 1, CI = 1, and CI > 1 were considered to indicate synergistic, additive, and antagonistic actions, respectively.

The cytotoxic activities of NPOx, Et-NPOx and B-NPOx were evaluated against monkey kidney epithelial cells (Vero cell line) using an alamar blue (Invitrogen, Life Technologies, Sacramento, CA. USA) viability assay. The cells were cultured in standard culture medium (MEM with 10% fetal bovine serum) in 24-well plates (7.5 × 10³ cells/well) to 80% confluence. Increasing concentrations of NPOx, Et-NPOx and B-NPOx (0.1–3 mM) in 5 μL of ethyl alcohol were added to each well. The medium was replaced after 24, 48 and 72 h of incubation (37°C, 5% CO₂ and 95% relative humidity) with 200 μL of fresh medium containing alamar blue, and the fluorescence of each well was measured using a Perkin Elmer LS 55 spectrofluorometer (Perking Elmer, Waltham, MA, USA). Cell viability (%) was measured relative to the control wells (cultured in medium alone) according to the following calculation: Cell viability (%) = (fluorescence_test × 100%)/fluorescence_control.

Tumor cells (5 × 10³) were plated in complete medium in the presence/absence of DMSO control or PLX4720 or PD0325901 inhibitors. Cells were cultured for 72 h, at which time the medium received 1 × AlamarBlue (10% v/v; Life Technologies). Cells were allowed to reduce AlamarBlue for ~4 h and fluorescence was measured at 560/590 nm using a PerkinElmer Envision XciteMultilabel plate reader. Results are expressed as relative viability and are calculated as a proportion of fluorescence counts of tumor cells in the presence of drugs vs. tumor cells in the presence of vehicle control.

Vegetative cells were diluted to approximately 2.5 × 10⁵ CFU ml⁻¹ in fresh Dulbecco’s modified Eagle’s medium (Gibco-Invitrogen, Carlsbad, CA) containing 10% (vol/vol) fetal bovine serum (HyClone, Logan, UT) and the various concentrations of CXCL10 or CTTC to be tested. Aliquots of 100 µl were placed in triplicate wells of a 96-well plate and incubated at 37°C with 5% CO₂ for approximately 4 h, unless otherwise noted, at which point alamarBlue dye (AbD Serotech, Oxford, United Kingdom) was added at a 1:10 dilution and allowed to reach visual saturation in an untreated control under protection from light. Active metabolism of the vegetative cells was quantified via the generation of a fluorescent signal from the reduction of the alamarBlue oxidation-reduction dye by measuring the fluorescence at 530-nm excitation and 590-nm emission wavelengths with a PerkinElmer Victor³ multilabel plate reader (PerkinElmer, Waltham, MA). The percentage of the control was calculated by comparison of the reading obtained with the chemokine-treated samples to that obtained with the corresponding untreated samples. Light microscopy was used for bacterial cell visualization. Camera control and image capture were performed with the QCapture pro-5.1 software as previously described (17 (link), 35 (link)).