E0.2 cells were maintained in DMEM supplemented with 10% FBS and 1% dual antibiotics. E0.2 cell line is a metastatic subclone of E0771 cell line generated in our laboratory [38 (link)]. MVT‐1 cells (derived from MMTV‐c‐Myc; MMTV‐VEGF bitransgenic mice) were obtained from Johnson [35 ] and were cultured as described previously [21 (link)]. The SUM159 cell line was kindly provided by S. Majumder (The Ohio State University) and cultured as described earlier [36 ]. The human breast carcinoma cell lines, MDA‐MB‐231 and MDA‐MB‐468, were obtained through ATCC. S100A7 overexpression or knockdown cells were generated from our previous studies [25 (link), 37 (link)]. PrestoBlue dye (Invitrogen, Eugene, OR, USA) was used to calculate cell viability. LPS and LPS blocker (polymyxin B, PMB) were purchased from Sigma‐Aldrich. All the cell lines were routinely checked for mycoplasma contamination and verified based on cell morphology. shRNAs targeting mouse Tlr4 (Locus ID 21898) were purchased from Origene and transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) into MVT1 cells. Breast tumor microarrays (TMAs; BR1002b) were purchased from US Biomax, Inc (Rockville, MD, USA). The clinicopathological details of TMA are available in Table S1 .
Prestoblue dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
PrestoBlue is a resazurin-based cell viability reagent that can be used to assess the metabolic activity of cells. It is a simple, fluorometric or colorimetric assay that provides a quantitative measure of cell proliferation and cytotoxicity.
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Lab products found in correlation
Synergy h1 microplate reader, by Agilent Technologies (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Genios, by Tecan (1 mentions)
Murine il 1β, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Mk 2206, by Adooq (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Yeast extract, by Merck Group (1 mentions)
Concentrated hydrochloric acid, by Merck Group (1 mentions)
Concentrated sulfuric acid, by Merck Group (1 mentions)
Peptone, by Merck Group (1 mentions)
Beef extract, by Merck Group (1 mentions)
1 kb plus dna ladder, by Thermo Fisher Scientific (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Loading dye, by Thermo Fisher Scientific (1 mentions)
Natural graphite flake, by Merck Group (1 mentions)
Potassium permanganate, by Merck Group (1 mentions)
Ciprofloxacin, by HiMedia (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Ceftriaxone, by HiMedia (1 mentions)
12 protocols using prestoblue dye
1
Metastatic Breast Cancer Cell Culture and Characterization
2
MTT and PrestoBlue Cytotoxicity Assays
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The peptides and NPP toxicity to cells were assessed using MTT cell viability assay. The concentration of peptide at 5µg was used to access cell viability using MTT because of efficient cellular uptake shown by flow cytometry. Moreover, the viability of NPP (5µg) treated HL60 cells was measured using the PrestoBlue dye (Invitrogen). The KG1a and K562 cells in 100µL IMDM supplemented with 20% and 10% FBS, respectively, were seeded in 96 wells plate at a density of 2.5×104 cells/well in triplicates and incubated for 24h. However, MO7e (supplemented with 10ng/mL SCF) and HL60 cells in 100µL of RPMI supplemented with 10% FBS were seeded and incubated at 37°C with 5% CO2 for 40h and 24h, respectively. Afterward, MTT was added and incubated for 2h and followed by DMSO addition for another 2h followed by absorbance measured by a spectrophotometer (BioTek Instruments, India) at 570nm, and 690nm (reference λ). Besides, PrestoBlue reagent, 10µL was added to each well contained nanoparticle-treated cells, followed by incubation for 10mins and finally read the absorbance at 570nm, and reference at 600nm.
3
PrestoBlue Viability Assay Protocol
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A 1:10 dilution of the 10× concentrated PrestoBlue dye (Invitrogen, Thermo Scientific, Karlsruhe, Germany) was prepared in serum-free RPMI medium. After the removal of media, 300 µL of the solution was added to the samples (24-well plate). Then, the cells were incubated for 45 min at 37 °C. Afterwards, 100 µL of the solution was transferred at least in two technical replicates to a 96-well plate. Finally, fluorescence was measured by a Tecan GENios plate reader (Tecan Deutschland GmbH, Mainz-Kastel, Germany) at a wavelength of 590 nm.
4
PDGF-BB and IL-1β Proliferation Assay
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Proliferation assays were performed as described previously70 . Briefly, cells were starved 24 h with α-MEM containing 1% FBS. Then, cells were seeded in 96-well plate with α-MEM containing murine PDGF-BB (5 ng ml−1, Peprotech) and 1% FBS, or α-MEM containing 10% FBS. Directly after, murine IL-1β (Peprotech) was added at concentrations ranging from 1 to 1,000 ng ml−1. For Akt and β-catenin inhibition experiments, MK-2206 (AdooQ Bioscience) or XAV-939 (AdooQ Bioscience) were added instead of IL-1β at a concentration of 10 μM. After 72 h, cell number was quantified using PrestoBlue dye (Invitrogen). Percentage proliferation increases were calculated over basal proliferation (medium with 1% FBS and no PDGF-BB).
5
Cytotoxicity Evaluation of Cisplatin and CBD
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Cell viability assays was performed using Presto Blue dye™ (ThermoFisher, Carlsbad, CA, USA) as per manufacturer’s instructions. Briefly, 2 × 103 cells were seeded per well in 90 μL of medium in 96-well plates and serum starved for 4 h before treatment. Cells were treated with increasing concentrations of cisplatin and CBD for 48 h. At the end of this period, Presto Blue™ solution was added to each well and incubated for 30 min. Fluorescence was measured at 570 nm and 600 nm using the BioRad (Hercules, CA, USA) microplate reader. Dose–response graphs were constructed, from which IC50 concentrations were determined for each cell line.
6
Quantifying Viable SVF Cells
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PrestoBlue dye was purchased from ThermoFisher (Cat#P50201) and used to quantify viable SVF cells at aforementioned timepoints. Absorbance was measured using an Infinite M200 Plate reader (Tecan Life Biosciences, Männedorf Switzerland). These results were then normalized to ASCs grown in D10 as a control.
7
Graphite Oxide Synthesis and Antibacterial Evaluation
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Natural graphite flakes, concentrated sulfuric acid (98%), potassium permanganate, 30% hydrogen peroxide, concentrated hydrochloric acid (98%) and ultrapure water were purchased from Sigma-Aldrich, Malaysia. All of the aqueous solutions were prepared in deionized water. Analytical grade absolute ethanol, NaCl, peptone, yeast extract, beef extract, agar were purchased from Merck, Germany. Antibacterial discs: azithromycin (AZM), gentamycin (GEN), ciprofloxacin (CIP), cefixime (CFM), amoxicillin (AMX), cotrimoxazole (COT), imipenem (IPM) and ceftriaxone (CTR) were bought from Himedia Laboratories, India. 6X loading dye, 1 Kb plus DNA ladder and Presto Blue dye were purchased from Thermo Fisher Scientific. Human serum and bacterial strains collected from urine were obtained from North East Medical College Hospital, Sylhet, Bangladesh (https://www.nemc.edu.bd/pathology-department/ ) following all the ethical conditions and legislations approved by the ethical committee with the Ref. No. NEMC/Sylhet/287/2013 [24 ].
8
Cytotoxicity Assay for Curcumin Compounds
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The cytotoxicity assays were performed using PrestoBlue dye as per the manufacturer’s instructions. Since the goal of these cytotoxicity assays was to demonstrate that the binding observed in the CBAs was not due to cell death, the protocol was modified to mimic that of a CBA. The cells were seeded at 30,000 cells per well in 96-well plates, incubated until they were 70–80% confluent, and then treated using different concentrations of CUR or CTCUR for 2h. CUR and CTCUR were dissolved in DMSO at a concentration of 0.1 M and diluted for the assays as required. Cell survival was monitored using PrestoBlue dye (Thermo Fisher Scientific, Waltham, MA, USA) per the manufacturer’s instructions, and the fluorescence was measured at 560 nm/590 nm (excitation/emission) using a Synergy H1 microplate reader and the Gen5 software (BioTek, Winooski, VT, USA). The percent change was calculated relative to the average for the control cells (no treatment).
9
Cell Stress Assay Protocol
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Chem stress assays were performed according to the manufacturer's instructions (ChemStress®, Valitacell Ltd). In brief, CHO or H2O2 evolved host cells were seeded into Valitacell ChemStress plates at 18,000 cells/well in 90 µl AstraZeneca proprietary medium supplemented with 6 mM l ‐glutamine. A control well was incubated with medium alone. Plates were incubated for 72 h in a static incubator at 36.5°C, 6% CO2. Following this, 10 µl of neat PrestoBlue dye (Thermo Fisher Scientific) was added to all wells before plates were mixed for 20 s and incubated for a further 30 min at 36.5°C, 6% CO2. Plates were analyzed using a PHERAstar plate reader (BMG LABTECH with preconfigured protocols (excitation 560 nm, emission 590 nm). Data were analyzed using the ValitaAPP software (Valitacell Ltd).
10
Presto Blue Dye Colorimetric Assay
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The direct killing assay was performed using Presto blue dye (Thermo Scientific) in a colorimetry based assay. Briefly, the compounds were added directly to S. typhi cells in a 96 well plate. The cells and the compounds were co-incubated in a 5% CO2 incubator for 24 h. After 24 h incubation, 20µl of Presto blue (Thermo scientific) was added directly to the culture media, and changes in color were determined through colorimetry.
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