Following the short-term and medium-term exposures, tissue viability test was carried out using the PrestoBlue assay. The PrestoBlue reagent (Biosource, Camarillo, CA) was added to the samples at a ratio of 9:1 (volume of cells and culture medium: volume of PrestoBlue reagent). The plates were then incubated for 60 minutes at 37°C and 5% CO2. Following incubation, triplicate 200-mL samples were placed into the wells of a 96-well plate, and the fluorescence intensity of each well was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a fluorescent plate reader (Infinite 200 PRO TECAN, Switzerland).
Prestoblue reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Italy, France, Denmark, Canada, Netherlands, Sweden, Belgium
PrestoBlue is a cell viability reagent that can be used to measure the metabolic activity of cells in culture. It is a resazurin-based solution that can be added directly to cells and incubated, after which the degree of fluorescence or absorbance is measured to determine cell viability.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Synergy ht, by Agilent Technologies (4 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Merck Group (3 mentions)
Mycoalert mycoplasma detection kit, by Lonza (3 mentions)
Polarstar omega, by BMG Labtech (3 mentions)
Prestoblue, by Thermo Fisher Scientific (3 mentions)
Sunrise elisa reader, by Tecan (3 mentions)
Delfia cell proliferation kit, by PerkinElmer (3 mentions)
Delfia dna fragmentation assay, by PerkinElmer (3 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (3 mentions)
Xmark microplate absorbance spectrophotometer, by Bio-Rad (2 mentions)
Spectramax m2, by Molecular Devices (2 mentions)
Synergy 2, by Agilent Technologies (2 mentions)
Mh broth, by Merck Group (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Cytation 5 plate reader imager, by Agilent Technologies (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
257 protocols using prestoblue reagent
1
Tissue Viability Assay using PrestoBlue
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Following the short-term and medium-term exposures, tissue viability test was carried out using the PrestoBlue assay. The PrestoBlue reagent (Biosource, Camarillo, CA) was added to the samples at a ratio of 9:1 (volume of cells and culture medium: volume of PrestoBlue reagent). The plates were then incubated for 60 minutes at 37°C and 5% CO2. Following incubation, triplicate 200-mL samples were placed into the wells of a 96-well plate, and the fluorescence intensity of each well was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a fluorescent plate reader (Infinite 200 PRO TECAN, Switzerland).
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Following the short-term and medium-term exposures, tissue viability test was carried out using the PrestoBlue assay. The PrestoBlue reagent (Biosource, Camarillo, CA) was added to the samples at a ratio of 9:1 (volume of cells and culture medium: volume of PrestoBlue reagent). The plates were then incubated for 60 minutes at 37°C and 5% CO2. Following incubation, triplicate 200-mL samples were placed into the wells of a 96-well plate, and the fluorescence intensity of each well was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a fluorescent plate reader (Infinite 200 PRO TECAN, Switzerland).
2
MSCs Growth on 3D Polymer Scaffolds
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The scaffolds were sized as 1 cm2 squared pieces, with a sterile scalpel (0.5 cm thickness), and sterilized by UV-rays exposure for 2 h, and finally pre-incubated for 2 h with complete medium in 24-wells plates.
HPCy-MSCs were seeded in concentration of 7 × 105 on PLA-10CaSi, PLA-5CaSi-5DCPD and PLA-10CaSi-10DCPD scaffolds and incubated in α-MEM complete medium (see above) at 37 °C and 5% CO2 enriched atmosphere; pure PLA scaffolds were used as control.
The same number of cells were plated on tissue culture treated 24-well plate as growth control.
Presto Blue reagent (Invitrogen, A13261, Carlsbad, CA, USA) has been used as metabolic assay for the evaluation of cell growth. Presto Blue reagent was diluted according to manufacturer’s instructions and added to the wells containing the scaffolds seeded with hPCy-MSCs or the empty scaffolds (negative control).
After incubation for 2 h at 37 °C and 5% CO2, the absorbance of resazurin dye was measured using a Multiskan GO (Thermo Fisher N10588, Waltham, MA, USA) spectrophotometer at 570–600 nm wavelength. Metabolic assays were performed at days 3, 7, 10, and 14.
HPCy-MSCs were seeded in concentration of 7 × 105 on PLA-10CaSi, PLA-5CaSi-5DCPD and PLA-10CaSi-10DCPD scaffolds and incubated in α-MEM complete medium (see above) at 37 °C and 5% CO2 enriched atmosphere; pure PLA scaffolds were used as control.
The same number of cells were plated on tissue culture treated 24-well plate as growth control.
Presto Blue reagent (Invitrogen, A13261, Carlsbad, CA, USA) has been used as metabolic assay for the evaluation of cell growth. Presto Blue reagent was diluted according to manufacturer’s instructions and added to the wells containing the scaffolds seeded with hPCy-MSCs or the empty scaffolds (negative control).
After incubation for 2 h at 37 °C and 5% CO2, the absorbance of resazurin dye was measured using a Multiskan GO (Thermo Fisher N10588, Waltham, MA, USA) spectrophotometer at 570–600 nm wavelength. Metabolic assays were performed at days 3, 7, 10, and 14.
3
Ferroptosis Sensitivity in TP53 Mutants
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H1299 cells expressing exogenous WT or mutant TP53 were seeded at 4 × 103 cells per well in a 96-well plate and allowed to grow for 24 h before treatment with 5 µM erastin, or a vehicle control (DMSO) along with 10 µg tetracycline for 24 h. Co-treatment with 10 µM ferrostatin-1, a ferroptosis inhibitor, was used as a negative control. Cell viability was assessed using PrestoBlue reagent (Thermo Fisher Scientific, Waltham, MA, USA) by adding 10 µL of PrestoBlue reagent to each followed by incubation at 37 °C for 20 min. Differences in fluorescence absorbance were measured using a Biotek Synergy HT (Biotek, Winooski, VT, USA) plate reader. Each assay included four technical replicates and was repeated at least three times. Differences in cell viability were determined by normalizing reductions in fluorescent absorbance relative to the vehicle control group for each cell line. To adjust for differences in size in the cells expressing endogenous WT or mutant TP53, cells were plated such that they were ~70% confluent on the day of treatment. Cells were treated with a vehicle control (DMSO) or 20 µM erastin for 24 h, and cell viability was assessed using PrestoBlue reagent as described above.
4
Assessing Cordycepin Cytotoxicity on CCA Cells
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The toxicity of cordycepin to CCA cell lines was measured by using PrestoBlue™ reagent (Invitrogen, Carlsbad, CA, USA). The CCA cell lines were cultured in 96-well plates with approximately 1 × 104 cells per well with a 2-fold serial dilution of cordycepin (Sigma-Aldrich, Darmstadt, Germany) between 3.125–400 µM, and incubated for 24 h. Then, the culture media was replaced with the PrestoBlue™ reagent (Invitrogen) at 100 µL per well. The reagent has the active ingredient called resazurin, which can be reduced by receiving electrons from the products of cellular respiration in living cells i.e., NADPH, FADH, FMNH, NADH and cytochromes. The reaction causes the change of resazurin to resorufin which turns the color from blue to pink which can be measured by recording the changes of absorbance at 570 nm (using 600 nm as a reference wavelength). The magnitude of color changes depends on the metabolic activity in living cells.
5
Quantitative Metabolic Assay with BIX01294 and GSK126
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Cells were cultured in a 24 well culture plate for 24 h and washed once with PBS followed by adding fresh medium containing 1 nM–10 μM of BIX01294 and/or GSK126 (in DMSO, final concentration 0.1% v/v) in triplicates for 72 h. Control cells were also cultured and treated simultaneously with the equivalent volume of DMSO at 0.1% finalconcentration (v/v). Cells were incubated for 72 h and PrestoBlue® reagent (Invitrogen) was added to a 1x final concentration followed by incubating the cells for further 3 h. Upon entering a living cell, PrestoBlue® reagent is reduced from resazurin, a blue compound with no intrinsic fluorescent value, to resorufin which is red in color and highly fluorescent. Conversion is proportional to the number of metabolically active cells and therefore can be measured quantitatively. The fluorescence intensity was then measured using xMarkTM Microplate Absorbance Spectrophotometer (Bio-Rad) with an excitation wavelength of 570 nm and emission wavelength of 600 nm.
6
Metabolic Activity Kinetics in Cell Cultures
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Total metabolic activity was analyzed at 48 h, 7 d, and 14 d for hGFs and at 48 h, 8 d, and 15 d for hBM-MSCs culture using Presto Blue reagent (Life Technologies, Carlsbad, CA, USA), following the manufacturer’s protocol, at 1 h of reagent incubation time.
7
Splenocyte Proliferation Assay for Leishmaniasis
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Spleens were individually homogenized in Roswell Park Memorial Institute (RPMI) 1640 medium and erythrocytes were lysed using lysis buffer (150 mM NH4Cl, 7 mM K2HCO3 and 0.01 mM EDTA). Cells were adjusted to 5 × 105 cells/well and cultured in sterile 96-well plates under specific stimulation with the total antigen (T-AG) of L. infantum promastigotes (10 μg/well) or under unspecific stimulation with concanavalin A (1 μg/well) as a positive control of proliferation. In addition, cells from all groups were cultured only with RPMI 1640 medium as negative controls. Plates were cultured in a humidified incubator at 37 °C under 5% CO2. Following 48 h of incubation, the plates were washed with PBS three times at 1000 rpm for 10 min, at 4 °C. Then, PrestoBlue reagent (Life Technologies, Carlsbad, CA, USA) was added to measure cellular proliferation (Sá-Nunes et al., 2009 (link), Hamalainen-Laanaya and Orloff, 2012 (link)). After 2 h, fluorescence was read with the excitation and emission wavelengths at 570 nm and 620 nm, respectively. The fluorescent levels of negative controls were subtracted from all samples. The PBS control group (uninfected, untreated) did not proliferate under stimulation with the T-Ag of L. (L.) infantum.
8
Evaluating DPMI-α Peptide Cytotoxicity
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T47D cells were cultured in phenol red-free RPMI supplemented with 2 % FCS, 100 U/mL penicillin and 100 ug/ml streptomycin. Cells were seeded at 8,000 cells/well in 96-well plates, incubated for 24 h, and then treated with the DPMI-α peptides at indicated doses. After 24 and 48 h PrestoBlue® reagent (LifeTech) was added to the media according to manufacturer’s instructions. Cells were incubated for a further 30 min and fluorescence read using an EnSpire® multimode plate reader (Perkin Elmer). Error bars represent standard error of the mean between duplicate samples.
9
Cell and Spheroid Viability Assays
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Cell viability was estimated after addition of PrestoBlue™ reagent (Life Technologies, Carlsbad, CA, USA) for 3 h, following the supplier protocol. For spheroid viability assays, the viability was estimated after addition of CellTiter‐Glo® 3D for 1 h, following the supplier protocol (Promega, Madison, USA).
10
Cell Viability Assay of Thymocytes and Splenocytes
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Thymocytes and splenocytes were seeded at a density of 0.1 × 106 cells/200 µl/ well in 96 well culture plates and incubated in a 5% CO2 incubator at 37 °C for 72 h. Cell viability was determined using Presto blue reagent (Life Technologies) following manufacturer’s protocol in a microplate reader (FLUOstar Omega, BMG).
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