Ezcount mtt cell assay kit

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the cytotoxicity of Aptamer R, T, J, and L using EZcount™ MTT cell assay kit (Himedia, Mumbai, India) in Vero cells as mentioned earlier^{21 (link)}. In brief, Vero cells were seeded in a 96 well plate at the density of approximately 10,000 cells per well. After reaching 80% confluency, cells were treated with different concentrations of aptamer R and T (1, 2, 3, and, 4 µM) and J and L (2, 4, 6, and, 8 µM) in triplicate form. 1X PBS was taken as a reagent control. After 22 h, plate was washed with 1X PBS and subsequently, 100 μl MTT reagent (1 mg/ml diluted in DMEM serum free media) (Himedia, Mumbai, India) was added to the cells and incubated for 2–3 h at 37 °C. Next, the media was removed and 100 μl of solubilization buffer was added followed by incubation at 37 °C for 15 min to dissolve the formazan crystals. Finally, the absorbance was measured at 570 nm using a multimode plate reader and the metabolically active cell percentage was compared with the control cells to determine the cytotoxicity^{21 (link)}.

The polydimethylsiloxane (PDMS) (SYLGARD 184 silicone elastomer and curing agent) used for device fabrication was purchased from Dow Corning Pvt. Ltd., Bangalore, Karnataka, India. The primary metal salt, Cobalt (II) Nitrate hexahydrate (Co(NO₃)₂.6H₂O), and linker, 2-Methylimidazole (2-MIM), were purchased from AVRA Pvt. Ltd., Bangalore, Karnataka, India. The secondary metal salt, Zinc Nitrate hexahydrate (Zn(NO₃)₂.6H₂O), metal salt (linker), Calcium chloride (CaCl₂), and biopolymer, sodium alginate, were purchased from NICE Chemicals Pvt. Ltd., Bangalore, Karnataka, India. The drug diclofenac sodium was purchased as tablets from CIPLA, and the tablets were crushed into a powdered form. The MTT assay kit (EZcount MTT Cell Assay Kit) was purchased from HiMedia Laboratories Pvt. Ltd., Bangalore, Karnataka, India. Dulbecco’s Modified Eagle Medium (DMEM), Dimethyl Sulfoxide (DMSO), and phosphate-buffered saline (PBS) buffer were purchased from Sigma Aldrich (India) Pvt. Ltd., Bangalore, Karnataka, India. MCF-7 breast cancer cells were acquired from the Department of Biotechnology, JAIN (Deemed-to-be-University), Karnataka, India.

The cytotoxicity of AAKi in Vero cells was determined using the EZcount MTT cell assay kit (Himedia, India) according to the manufacturer’s protocol. Approximately 20,000 cells were plated in a 96-well plate 1 day before the experiment. Upon reaching 80% confluence, the cells were treated with different concentrations of the inhibitor for 15 h, and DMSO was used as the reagent control. The percentage of metabolically active cells was compared with the control cells, and cellular cytotoxicity was determined as described previously (37 (link)).

The assay was performed to evaluate the cytotoxic properties of ACL extract on murine splenocytes and macrophages. The splenocyte and macrophage cells were collected from Swiss albino mice [33 ]. Swiss albino mouse was sacrificed under mild ether anesthesia and the spleen was aseptically removed from the body and washed thrice (1000 rpm) with RPMI-1640 and splenocytes suspension was prepared and resuspended in 0.16 M NH₄Cl (in 0.17 M Tris; pH 7.2) to remove any trace of erythrocytes. After 5 min, the reaction was stopped using chilled RPMI-1640 and the cells were washed as previous. Peritoneal exudate macrophages were collected by washing the mouse peritoneal region with RPMI-1640. Cell suspension (2×10⁶ cells/ml) was prepared with penicillin (50 U/ml), streptomycin (50 U/ml), nystatin (50 U/ml) and fetal bovine serum (FBS, 10%) in RPMI-1640 medium. The cell suspension (100 μl) was added with 100 μl of different concentrations (0–200 μg/ml) of ACL (dissolved in RPMI-1640) to the wells of 96-well plate. The plates were then covered and incubated under 5% CO2 and humidified atmosphere of 90% air at 37°C temperature for 48 h. The cytotoxicity assay was performed according to the manufacturer’s instructions of EZcount^™ MTT Cell Assay Kit (HiMedia).

To determine the cytotoxicity of MBZM-N-IBT, an MTT assay was performed using the EZcount MTT cell assay kit (Himedia, Mumbai, India) in RAW 264.7 cells according to the manufacturer’s protocol. Approximately 10,000 numbers of cells were seeded in 96-well plates 24 h before the experiment. At 80% confluence, the cells were treated with increasing concentrations of compound. For this assay, at 12 h post-treatment, 10 μL MTT reagent (5 mg/mL) was added to the cells and incubated for 1 h at 37°C. Next, the medium was removed, and 100 μL of solubilization buffer was added followed by incubation at 37°C for 15 min to dissolve the formazan crystals. Finally, at 570 nm, the absorbance was measured using a multimode plate reader (Perkin-Elmer, MA) to determine the cellular cytotoxicity (56 (link)).

MTT assay was performed to assess cytotoxicity of 17-AAG and Z-VAD-FMK using EZcount™ MTT cell assay kit (Himedia Laboratories Pvt. Ltd., Mumbai, India) according to the manufacturer’s instructions. Briefly, the Raw264.7 cells were seeded in 96 well plate at a density of 5 × 10³ cells per well before drug treatment. The cells were then washed in 1× PBS and incubated with different concentrations of drugs in triplicate. The DMSO was taken as solvent control. After 24 h of treatment, the cells were incubated with the MTT reagent to a final concentration of 10% of the total volume. Then, the cells were incubated for 2 h in the incubator for the formation of visible crystals. Later, the media was removed carefully and 100 µL of solubilization solution was added per well followed by incubation for 15 min at room temperature (RT). The absorbance of the solution was taken at 550 nm by Microplate Reader (Bio-Rad, Hercules, CA, USA). Next, the percent viable cells were calculated in comparison to the control cells of the same plate in triplicate.