NIH3T3 mouse fibroblasts (CRL-1658), CaCo2 cells human colorectal adenocarcinoma (HTB-3), and the N2a neuroblastoma cell line (CCL-131) were obtained from the American Type Culture Collection and were validated by genomic PCR. Primary human fibroblasts (GM01651) were from Coriell Repository. Cells were cultured to full confluence in a 37 °C incubator with 5% CO2 in complete Dulbecco’s modified Eagle’s medium (DMEM, Sigma #D5030) supplemented with 10% heat-inactivated newborn calf serum (HyClone #SH30401.02HI), 1% penicillin/streptomycin/fungizone (Invitrogen, #10072322). All the cell lines were tested for mycoplasma contamination using DNA staining protocol with Hoechst 33258 dye and none of them was found positive. Knock-down of Ncor1 was done using lentiviral mediated shRNA (SHCLNG-NM_011308) from the Sigma Mission library following standard procedures. The efficiency of knock-down was tested by immunoblot. Cell viability was measured using the CellTiter-Blue kit (Promega, #G9241) 24 h after the addition of the different stressors according to the manufacturer’s instructions. All cell lines were validated by genomic PCR.
Celltiter blue kit
Manufactured by Promega
Sourced in United States
The CellTiter-Blue kit is a cell viability assay used to measure the metabolic activity of cells. It utilizes a redox dye that changes color in response to the reduction of metabolites by viable cells. The assay allows for the quantification of cell proliferation and cytotoxicity in a simple, homogeneous format.
Automatically generated - may contain errors
Lab products found in correlation
Gm01651, by Coriell Institute for Medical Research (1 mentions)
Penicillin streptomycin fungizone, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Caco 2 cell, by American Type Culture Collection (1 mentions)
Crl 1658, by American Type Culture Collection (1 mentions)
Fluostar omega plate reader, by BMG Labtech (1 mentions)
Int bcip brown kit, by Sangon (1 mentions)
Synergy neo2, by Agilent Technologies (1 mentions)
Streptomycin sulfate, by Corning (1 mentions)
Amphotericin b, by Corning (1 mentions)
Hexamethylene diisocyanate hdi, by Merck Group (1 mentions)
Penicillin g, by Corning (1 mentions)
Anhydrous n n dimethylformamide dmf, by Thermo Fisher Scientific (1 mentions)
1 ethyl 3 3 dimethylaminopropyl carbodiimide, by Oakwood Chemical (1 mentions)
N n dicyclohexylcarbodiimide, by Merck Group (1 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Corning (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BioLegend (1 mentions)
Cell titer blue, by Agilent Technologies (1 mentions)
25 protocols using celltiter blue kit
1
Profiling Cell Viability Across Lines
2
Evaluating PRLR Variant Cell Viability
3
Measuring Cellular Metabolic Activity
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To analyze cellular metabolic function we utilized Cell Titer-Blue Kit from Promega. We plated 1000 RKO cells that had been stably transfected with MECP2-WT or K171Q mutant into black fluorescence compatible 96-well microtiter plates. Cells were allowed to grow in culture for 3 days. On the final day in culture, Cell Titer-Blue reagent was added to each well and incubated for 2 hours at 37°C. Cells were then analyzed for fluorescence at 560/590nm emission using a BioTek plate reader and Gen5 software. The data were quantitated and compiled from four independent experiments.
4
Protein Analysis Methods in Cell Stress
5
Cell Viability Assay with Doxycycline-Induced Flp-In T-Rex 293 Cells
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Flp-In T-Rex 293 cells were induced with doxycycline (1 μg/ml) for 24 hours, seeded at densities of 2000 to 20,000 cells per 100 μl, and treated with the indicated concentrations of CPT in the absence of doxycycline for 72 hours. Cell viability was measured using the CellTiter-Blue kit (Promega). The CellTiter-Blue reagent (20 μl) was mixed with the cells and incubated at 37°C for 1 hour. Fluorescence intensity was measured at an excitation/emission wavelength of 544/590 ± 10 nm using a FLUOstar Omega plate reader (BMG Labtech). Viability of untreated cells was set to 1, and error bars represent SE from three independent biological repeats.
6
Evaluating Cell Viability under ACR
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U87 and U251 cells were subjected to transfection and ACR treatment as described above. Cells were then cultured in DMEM in 96-well plates in triplicates. Cell survival was measured with the CellTiter-Blue kit (G8080; Promega, Madison, WI, USA) according to the manufacturer's instructions. Fluorescence was detected at 560 nm using a microplate reader.
7
Monitoring mESC Viability and Alkaline Phosphatase
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mESCs were seeded into 96 well plates in growth medium for 0 h, 24 h, 48 h, 72 h, and 96 h, respectively. CellTiter-Blue kit (Promega, Cat. #G8081) was used to measure cell viability at different time points. Fluorometric signals were recorded by a microplate reader (BioTek, Synergy NEO2).
mESCs were fixed with 1% paraformaldehyde before alkaline phosphatase staining. The activity of alkaline phosphatases in mESCs was detected by INT/BCIP (Brown) kit (Sangon Biotech, Cat. #C500033) following the manufacturer’s instructions. NIH3T3 cells were used as negative controls.
mESCs were fixed with 1% paraformaldehyde before alkaline phosphatase staining. The activity of alkaline phosphatases in mESCs was detected by INT/BCIP (Brown) kit (Sangon Biotech, Cat. #C500033) following the manufacturer’s instructions. NIH3T3 cells were used as negative controls.
8
Evaluating Ferroptosis Inhibition in Cu-Cy Mediated MWDT
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5 × 105 cells/well were implanted into a 6-well plate. After incubation at 37 °C for 24 h, fresh culture media with or without Cu-Cy was added respectively. After incubating for 24 h in an incubator, the MW treatment was carried out. Cell viability was determined using Cell TiTER-Blue® kit (Promega Co.Ltd). Briefly, 200 μL of Cell Titer-Blue reagent was added to each well and cultured in an incubator for 2 h. Fluorescence at 560/590 nm was determined using a microplate reader. The cell viability of experimental samples was calculated by comparing their fluorescence intensities with that of the control group. Three different iron death inhibitors, 5 mM N-Acetylcysteine (NAC), 100 μM deferoxamine (DFO), and 1 μM ferrostatin-1 (Fer-1) were used to investigate the effect of ferroptosis inhibition on Cu-Cy (20 μg/mL) mediated MWDT.
9
Synthesis and Characterization of PEG-HDI Hydrogels
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Boc-L-Serine and anhydrous N,N-dimethylformamide (DMF) were purchased from Acros. Hexamethylene diisocyanate (HDI) and N,N-dicyclohexyl carbodiimide (DCC) were bought from Sigma-Aldrich. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide was obtained from Oakwood Chemical. 1,3-Propanediol, 4-(dimethyl amino) pyridine (DMAP) and poly(ethylene glycol) mono methyl ether (MPEG) were purchased from Alfa Aesar. The HPLC grade tetrahydrofuran (THF), dichloromethane (DCM), ethyl acetate (EtOAc), hexanes and anhydrous diethyl ether were purchased from Pharmco Aaper. The dialysis tubing was purchased from VWR (cutoff MW = 3.5 kD, Spectra/Por). The Dulbecco’s Modification of Eagle’s Medium (DMEM, both with and without L-glutamine), the penicillin G, the streptomycin sulfate, the amphotericin B and the L-glutamine were all purchased from Cellgro. Fetal bovine serum (FBS) was purchased from Life Technologies. CE was obtained from Worthington Biochemical Corporation. The LIVE/DEAD viability/cytotoxicity kit was purchased from Molecular Probes. The Cell-Titer Blue kit was purchased from Promega. The rat monoclonal antibody ED1 (anti-CD68) was purchased from Abcam. The goat anti-rat secondary antibody conjugated with Alexa594 was purchased from Life Technologies.
10
Evaluation of Curcumin Nanoformulation Cytotoxicity
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Murine subcutaneous connective tissue fibroblasts (L929) were seeded at 10 × 103 cells per well of 96-well plates in triplicates for 24 h. Then, the respective complete culture media were exchanged for serum-free media only (serving as the no-treatment cell culture negative control, −ve ctrl), or containing several two-fold serial dilutions of free CUR dissolved in hydro-alcoholic solution (CUR-Sol), compared with empty δ-T3 and Tph NE vehicles and corresponding CUR-loaded prototype NE formulations. Finally, after 48 h of co-incubation, total L929 cell viability was determined using CellTiter Blue® Kit (Promega, Madison, WI, USA), after washing twice with Hank’s Balanced Saline solution, pH 7.4 (HBSS) and reading sample plate fluorescence (λEx = 480 nm/λEm = 530 nm), using Synergy 2 Biotek fluorescence plate reader (Biotek instruments Co., Winooski, VT, USA), according to the manufacturer’s instructions [19 (link),32 (link)].
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