To evaluate the stability of HGNs and PEG-HGNs in cell culture media at 0.125 mg mL−1, the BCA assay (ThermoFisher, USA) was performed as mentioned before to evaluate the total amount of proteins adsorbed on them. Moreover, their aggregation state was assessed by TEM and by Zeta potential analysis. Blue Cell viability assay ® (Abnova, USA) was employed to determine cell viability of MSCs under the effect of HGNs and PEG-HGNs [34 (link)]. To analyze the effects of HGNs and PEG-HGNs in cell cycle and in DNA damage, the distribution of cell cycle phases after NPs treatment was assessed by flow cytometry [34 (link)].
Blue cell viability assay
Manufactured by Abnova
Sourced in United States, United Kingdom
The Blue Cell Viability assay is a laboratory product that measures the viability of cells. It provides a quantitative assessment of the number of living cells in a sample.
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Lab products found in correlation
Multimode synergy ht microplate reader, by Agilent Technologies (2 mentions)
Synergy ht microplate reader, by Agilent Technologies (2 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Varioskan lux, by Thermo Fisher Scientific (1 mentions)
Human transferrin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Sodium selenite, by Merck Group (1 mentions)
Penicillin streptomycin amphotericin b, by Biowest (1 mentions)
Dmem ham s f12 medium, by Biowest (1 mentions)
Stable glutamine, by Biowest (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Dichloromethane, by Merck Group (1 mentions)
Mowiol 4 88, by Merck Group (1 mentions)
Antibiotics antimycotics, by Biowest (1 mentions)
Nhdf neo, by Lonza (1 mentions)
Rosmarinic acid, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Tween 80, by Merck Group (1 mentions)
8 protocols using blue cell viability assay
1
Stability and Biocompatibility of HGNs
2
Cell Viability Assay of Self-Assemblies
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The effect of self-assemblies in cell metabolism was determined by the Blue Cell Viability assay (Abnova, Taiwan), which is based on the reduction of a dye to a fluorescent compound mediated by metabolically active cells. For that purpose, cells were incubated with self-assemblies (0.01–0.4 mg mL−1) for 24 h. Then, the reagent was added (10%) and incubated for 4 h. After that, fluorescence was read at 535/590 nm (ex/em) in a Multi-mode Synergy HT Microplate Reader (Biotek, US). Cell viability was determined by the interpolation of the emission data obtained from the treated samples and the control ones, which were assigned with 100% viability.
3
Chondrogenic ATDC-5 Cell Viability Assay
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The mouse chondrogenic cell line ATDC-5 was kindly gifted by Dr. Oreste Gualillo (University of Santiago de Compostela, La Coruña, Spain). Cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM)–Ham’s F-12 medium (Biowest, Nuaillé, France) supplemented with 5% FBS (Gibco, UK), 10 μg/mL human transferrin (Sigma-Aldrich, St. Louis, USA), 3 × 10−8 M sodium selenite (Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin-streptomycin-amphotericin B (Biowest, Nuaillé, France), and 1% stable glutamine (Biowest, Nuaillé, France) in a humidified atmosphere of 5% CO2 at 37 °C.
The viability of cells was assessed using the Blue Cell Viability Assay (Abnova, Taipei, Taiwan). In brief, cells (104 cells/well) were cultured in 96-well plates and treated with onion extracts (OE) (1.9–62.5 µg/mL) for 24 h at 37 °C. After that, the reagent was added in accordance with the manufacturer’s instructions (10%; incubation of 4 h at 37 °C and 5% CO2). Metabolically active cells were able to reduce the dye, and the fluorescence generated was calculated in a microplate reader (Varioskan Lux, Thermo Fisher, Waltham, MA, USA) at 530 nm excitation and 590 nm emission wavelengths. Viability percentages were determined by linear interpolation of data considering control samples (non-treated cells) as 100% of viability.
The viability of cells was assessed using the Blue Cell Viability Assay (Abnova, Taipei, Taiwan). In brief, cells (104 cells/well) were cultured in 96-well plates and treated with onion extracts (OE) (1.9–62.5 µg/mL) for 24 h at 37 °C. After that, the reagent was added in accordance with the manufacturer’s instructions (10%; incubation of 4 h at 37 °C and 5% CO2). Metabolically active cells were able to reduce the dye, and the fluorescence generated was calculated in a microplate reader (Varioskan Lux, Thermo Fisher, Waltham, MA, USA) at 530 nm excitation and 590 nm emission wavelengths. Viability percentages were determined by linear interpolation of data considering control samples (non-treated cells) as 100% of viability.
4
Cytotoxicity Evaluation of PLGA Nanoparticles
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PLGA-Amine terminated/PLGA-NH2 (50:50) was purchased from GenoChem World SL (Valencia, Spain). Dichloromethane (DCM, >99.8%), dimethyl sulfoxide (DMSO, >99%), Mowiol® 4–88, Tween® 20, phosphate-buffered saline (PBS), dihydrorhodamine 123 (DHR123), protoporphyrin IX (PpIX), indocyanine green (ICG), and potassium chloride (KCl, 99.0–100.5%) were purchased from Sigma-Aldrich (Darmstadt, Germany). Tryptone soy agar (TSA) was purchased from Laboratorios Conda-Pronadisa SA (Madrid, Spain). Tryptone soy broth (TSB) was purchased from VWR Chemicals. S. aureus ATCC 29213 was obtained from Ielab (Alicante, Spain). Human dermal fibroblasts (NHDF-Neo, Lonza, Basel, Switzerland) were used in order to evaluate the potential cytotoxicity of PpIX and the synthetized particles. High-glucose Dulbecco’s modified Eagle’s medium (DMEM; DMEM w/stable glutamine) and antibiotics−antimycotics (60 μg/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B) were supplied by Biowest (Nuaille, France). The medium was supplemented with 10% (v/v) fetal bovine serum (FBS) from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). The Blue® Cell Viability Assay, used to evaluate the dose-dependent cytotoxicity, was purchased from Abnova (Taipei, Taiwan).
5
Onion Extract Cytotoxicity Screening
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Extracts from the onion waste were diluted in a supplemented cell culture medium to the final concentration of 1 mg/mL. For cytotoxicity screening assays, the cells were seeded in 96-well plates at a density of 4 × 103 cells/well. The culture medium was replaced with a medium containing onion extracts and the cells were incubated for 24, 48 and 72 h. The cells were washed twice with PBS after treatment and the Blue Cell Viability Assay (Abnova, Taipei, Taiwan) was developed by adding 10% of the reagent to the supplemented medium. After 3 h of incubation, viability was evaluated by fluorescence reading in a microplate reader (Multimode Synergy HT Microplate Reader; Biotek, Shoreline, WA 98133, USA) at 530 nm excitation and 590 nm emission wavelengths. Cell viability was calculated by linear interpolation of the fluorescence data from the treated cells versus the not treated sample (control sample containing 1% ethanol, 100% viability). The IC50 value indicates the concentration of the compound is capable of reducing cell viability to 50%. This value was calculated in all conditions tested and was chosen for subsequent assays.
6
Antimicrobial and Cytotoxicity Evaluation
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Carvacrol (CRV), cinnamaldehyde (CIN), thymol (THY), Squalene, Rosmarinic acid, β-Caryophyllene, Calcofluor White Stain, phorbol 12-myristate 13-acetate (PMA), and Tween® 80 were purchased from Sigma-Aldrich (St. Louis, MO, USA), while Eugenol was supplied by Acros Organics (Gell, Belgium). Tryptone soy broth (TSB) and agar (TSA) were obtained from Conda-Pronadisa (Madrid, Spain) and S. aureus (ATCC 25923) from Ielab (Alicante, Spain). Regarding cell lines, human dermal fibroblasts were purchased from Lonza (Bornem, Belgium), and THP1 human monocytes (ATCC TIB-202) from LGC Standards (Barcelona, Spain), while human keratinocytes were kindly gifted by Dr Pilar Martín-Duque. High-glucose DMEM (DMEM w/stable glutamine), RPMI 1640 w/stable glutamine, and antibiotic-antimycotic (60 µg/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin B) were supplied by Biowest (CEDEX, France). Cell culture reagents, such as fetal bovine serum (FBS), HEPES, nonessential amino acids, 2-mercaptoethanol 50 mM and sodium pyruvate 100 mM, were obtained from Gibco (Manchester, UK), and the Blue Cell Viability assay from Abnova (Aachen, Germany).
7
Cell Viability Assessment of BNC-Nanogels
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The blue cell
viability assay (Abnova, Taiwan) was employed to determine the viability
related to cell metabolism after cell culture treatment with BNC-nanogels,
BNCs, empty nanogels, and bupivacaine hydrochloride solution (0.01–0.5
mg/mL) for 24 h. The reagent was added to the cell cultures (10%)
and incubated for 4 h (37 °C, 5% CO2). Then, the emitted
fluorescence was read (535/590 nm ex/em) in a Synergy HT microplate
reader (Biotek). Control samples to evaluate the potential interference
of the BNCs and BNC-nanogels in the assays were also assayed. Viability
percentages were calculated by the interpolation of the emitted fluorescence
from the treated samples and control samples (control samples = 100%
viability).
viability assay (Abnova, Taiwan) was employed to determine the viability
related to cell metabolism after cell culture treatment with BNC-nanogels,
BNCs, empty nanogels, and bupivacaine hydrochloride solution (0.01–0.5
mg/mL) for 24 h. The reagent was added to the cell cultures (10%)
and incubated for 4 h (37 °C, 5% CO2). Then, the emitted
fluorescence was read (535/590 nm ex/em) in a Synergy HT microplate
reader (Biotek). Control samples to evaluate the potential interference
of the BNCs and BNC-nanogels in the assays were also assayed. Viability
percentages were calculated by the interpolation of the emitted fluorescence
from the treated samples and control samples (control samples = 100%
viability).
8
Blue Cell Viability Assay for Metabolic Effects
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Blue Cell Viability assay (Abnova, Taiwan), which is based on the reduction of a dye to a fluorescent compound mediated by metabolically active cells, was used to determine the effects of our hybrid thermoresponsive nanoparticles on cell metabolism after incubation (0.02-2 mg/mL) for 24 h. Then, the reagent was added to the cells (10%) and incubated for 4 h (37 ºC, 5% CO 2 ). After incubation, fluorescence was read (535/590 nm ex/em) in a Synergy HT Microplate Reader (Biotek, US) and viability was calculated by interpolation of the emission data displayed by the treated samples and the control ones (untreated samples = 100% viability).
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