Alamarblue cell viability assay

The alamarBlue cell viability assay (Bio-Rad Laboratories) was performed against selected cell lines, including the human lung cancer cell line A549, the human cervical cancer cell line HeLa, and the human colon cancer cell line HCT116. These cells were cultured in Dulbecco’s modified Eagle’s essential medium (DMEM; Gibco, New York, NY, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 50 U/mL streptomycin (Gibco, New York, NY, USA) at 37°C and 5% CO₂. In 96-well plates, 10 × 10³ cells were seeded per well for overnight incubation and treated with different concentrations of compounds 1 to 3 for 48 h. Each concentration was performed in triplicate, and the 50% inhibitory concentration (IC₅₀) value was analyzed using GraphPad Prism software.

A test procedure was developed to test effect of CPP_AIF on the barrier function on SkinEthic™ reconstructed human epidermis (RHE) based on the relevant procedure mentioned in OECD TG 439. This testing procedure involved topical application of CPP_AIF onto surface of the epidermis model for 1 h. After 1 h of exposure, CPP_AIF was washed by PBS from the surface followed by application of detergent solution (1% Triton X-100) onto surface of the tissue for another 2 h. Then, washed the detergent solution and AlamarBlue cell viability assay (BUF012B, Bio-Rad, Hercules, CA, USA) was performed for assessment of cell viability. Cell viability of treated models was normalized to the negative control, which was set to 100%.

Cellular viability was assessed for hASCs using an AlamarBlue Cell
Viability Assay (Biorad, U.K.). hASCs were seeded (2 × 10⁴ cells/gel) on the top of the gels (10 mM). After predetermined
culture times, the medium was carefully aspirated, and the hydrogels
were rinsed with 20% (v/v) AlamarBlue Cell Viability Assay solution
in α-MEM for 5 h at 37 °C with 5% CO₂. Following
incubation with the reagent, aliquots (100 μL) were placed into
black 96-well plates, and the fluorescence was measured using a plate
reader (Synergy, Bio-Tek, USA) with an excitation wavelength of 550
nm and an emission wavelength of 590 nm. Cells cultured on tissue
culture polystyrene (TCPS) were used as controls, and acellular gels
were used as blank (to adjust for background fluorescence).

To determine the IC50s of Ara‐C and clofarabine, an alamarBlue cell viability assay (Bio‐Rad Laboratories, Hercules, CA) was performed; 1 − 4 × 10⁵ cells were plated into a 96‐well flat‐bottom plate and cultured in triplicate in the absence or presence of seven concentrations of each drug for 68 h. After a 6‐h additional incubation with alamarBlue, the absorbance at 570 nm was monitored by a microplate spectrophotometer using 600 nm as a reference wavelength. Cell survival was calculated by expressing the ratio of the optical density of treated wells to that of untreated wells as a percentage. The concentration of agent required to reduce the viability of treated cells to 50% of untreated cells was calculated, and the median of three independent assays was determined as IC50. For detection of apoptosis, cells were cultured in the absence or presence of Ara‐C or clofarabine for 48 h, stained with FITC‐conjugated Annexin V and 7‐aminoactinomycin D (7AAD) (MBL, Nagoya, Japan), and analyzed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA).

Macrophage inflammatory assay were carried out to evaluate whether CPP_AIF displayed anti-inflammation potential [22 (link),23 (link),24 (link)]. In this experiment, macrophage (Raw264.7) cells were seeded in 96-well plates (5 × 10⁵ cells/mL) and allowed to attach overnight. After attachment, cells were incubated with various concentrations of CPP_AIF for 1 h and followed by stimulation with 1 μg/mL of LPS (lipopolysaccharide). No LPS-added cells were considered as control groups. After incubation, the amount of TNF-α and IL-6 in the medium were analyzed by enzyme linked immunosorbent assay (# KHC3011 and #EH2IL6, Thermo Fisher, Waltham, MA, USA). Cell viability was measured by AlamarBlue cell viability assay (BUF012B, Bio-Rad, Hercules, CA, USA).

Fifty percent inhibitory concentration (IC50) values of prednisolone (Pred), dexamethasone (Dex), vincristine (VCR), daunorubicin (DNR), L-asparaginase (L-Asp), cytarabine (AraC), methotrexate (MTX), mercaptopurine (6MP), and mafosfamide [Maf; active metabolite of cyclophosphamide (CY)] were determined using the alamarBlue cell viability assay (Bio-Rad Laboratories, Hercules, CA) as previously reported [17 (link)]. Cells (1–4 × 10⁵) were placed onto 96-well flat bottom plates in the presence or absence of seven separate concentrations of each drug in triplicate. The cells were cultured for 44 h to determine the DNR, VCR and CY (Maf) sensitivities and for 68 h to determine Pred, Dex, L‐Asp, MTX, and 6MP; 20 µL of alamarBlue was then added. After incubation for an additional 6 h in the presence of alamarBlue, the optimal density was read on a spectrophotometer at 570 nm using 600 nm as a reference wavelength. Cell viability was calculated by the ratio of the optical density of the treated wells to that of the untreated wells as a percentage. The concentration of each agent required to reduce the viability of the treated cells to 50% of the untreated cells (IC50 value) was calculated and the median IC50 value of three independent assays was determined.