Live dead cell assay kit

Standard ZEA (HPLC grade, 99% pure), caspase-3 assay kit, rhodamine 123, 4′,6-diamidino-2-phenylindole (DAPI), dichloro-dihydro-fluorescein diacetate (DCFH-DA), and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) were received from Sigma-Aldrich (Bengaluru, India). The live/dead cell assay kit was from Invitrogen Molecular Probes (Bengaluru, India). The Dulbecco's phosphate-buffered saline pH 7.4 (DPBS), antibiotic solution (streptomycin and penicillin), fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), and plasticware were obtained from HiMedia (Mumbai, India). Acetonitrile, methanol, dimethyl sulfoxide (DMSO), distilled water, and other chemicals of superior grade were bought from Merck Millipore Corporation (Bengaluru, India).

For cell viability analysis, the encapsulated cells in the constructs were evaluated using a Live/Dead Cell assay kit (Molecular Probes, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions for 7 days. All the constructs were washed with PBS (phosphate buffered saline, Gibco, Gaithersburg, MD, USA), moved to another 24 well plate. The samples were stained for 20 min in the dark and washed 3 times in PBS. Live and dead cells were observed using fluorescence microscopy (Nikon, Tokyo, Japan). Furthermore, the cell-laden constructs were cut on 2.4 × 2.4 mm² size and then analyzed with WST-1 (Life Technologies, Carlsbad, CA, USA) to analyze the cell proliferation for 4 days. Briefly, the constructs were washed with PBS, moved to another 48 well plate. In the next, 40 μL of WST-1 reagent and 400 μL of serum free medium were added in each well and then incubated in the dark for 40 min at 37℃. After incubation, 100 μL of the incubated medium was transferred to a 96 well plate, and the absorbance at 450 nm was immediately measured using a microplate spectrophotometer (Bio-Rad, Hercules, CA, USA). The cell viability was examined after incubation for five samples per each group, and the WST-1 reagent with serum free medium was used as the blank control.

Doxorubicin hydrochloride (DOX HCl) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Agarose was purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS), Dulbecco's phosphate buffered saline (PBS), penicillin/streptomycin, and trypsin/EDTA were obtained from Gibco (Carlsbad, CA, USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Hyclone (Logan, UT, USA). Live/Dead Cell Assay Kit, Quant-iT PicoGreen dsDNA Reagent and Kits, and the AlamarBlue Cell Viability Reagent were purchased from Invitrogen (Carlsbad, CA, USA). Complete protease inhibitor cocktail was purchased form Roche (Mannheim, Germany). Anti-GAPDH antibody and anti-CD44 antibody were purchased from Abcam (Cambridge, UK). Goat anti-rabbit IgG-HRP and the AnnexinV-FITC Apoptosis Detection Kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The Cell Cycle Detection Kit, the BCA Protein Assay Kit were purchased from Beyotime (Nanjing, China). The human breast adenocarcinoma cell line, MCF-7, was purchased from the Shanghai Institute of Cell Biology (Shanghai, China).