To evaluate cell membrane integrity, the cellular release in the supernatant of cytoplasmic lactate dehydrogenase (LDH) was assessed using the CytoTox-96™ Homogeneous Membrane Integrity Assay (Promega, Charbonnières-les-Bains, France) according to the manufacturer’s instructions. The optical density of the samples was determined using a microplate reader (Multiskan RC; Thermolabsystems, Helsinki, Finland) set to 450 nm. Three independent experiments were performed, each in quadruplicate and the activity of the released LDH was reported to that of negative control cells (incubated without nanoparticles). A positive control consisted of the maximal cellular LDH released after cells lysis.
Multiskan rc
Manufactured by Thermo Fisher Scientific
Sourced in Finland, United States
The Multiskan RC is a multi-channel absorbance microplate reader designed for general laboratory use. It measures absorbance at multiple wavelengths to quantify the concentration of samples in microplates. The device provides reliable and reproducible results for a variety of applications.
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Lab products found in correlation
Cytotox 96 homogeneous membrane integrity assay, by Promega (3 mentions)
Quantikine mouse tnf α immunoassay, by R&D Systems (3 mentions)
Ascent software v2, by Thermo Fisher Scientific (2 mentions)
Cell proliferation kit 1, by Roche (2 mentions)
Xtt assay, by Sartorius (2 mentions)
Quantikine human il 8 immunoassay, by R&D Systems (2 mentions)
Ortho nitrophenyl β galactoside onpg, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ldh cytotoxicity detection kit, by Merck Group (1 mentions)
96 well plate, by Corning (1 mentions)
Bapta, by Merck Group (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Db100b, by R&D Systems (1 mentions)
Quantikine elisa kit, by R&D Systems (1 mentions)
Elisa kits for tnf, by BioLegend (1 mentions)
Phenol red, by Merck Group (1 mentions)
Dmem without phenol red, by PAN Biotech (1 mentions)
27 protocols using multiskan rc
1
Evaluating Cell Membrane Integrity via LDH Release
2
In Vitro 5-ALA PDT Cytotoxicity
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5000 cells of each of the cell lines were seeded in a 96-well plate (Corning, Somerville, MA, USA) and treated with the above-mentioned in vitro 5-ALA PDT protocol, followed by recuperation of the media at 24 h post-illumination. Cells treated with 2% Triton X-100 (Sigma-Aldrich, Saint-Louis, MO, USA) for 2 h were considered as the positive control. The media was used immediately to analyze cytotoxicity by using a Cytotoxicity Detection (LDH) kit (Roche, Sigma Aldrich, Saint Louis, MO, USA) according to the manufacturer’s instructions. The measurements were done using a UV spectrophotometer (Multiskan RC, Thermo Fischer Scientific, Waltham, MA, USA) at 492 nm and was powered by Ascent™Software v2.06 (Thermo Fisher Scientific, Waltham, MA, USA). The cytotoxicity was represented as Relative Cytotoxicity (%) = ((Sample value − Non-Treated Control)/(Positive Control − Non-Treated Control)) × 100.
3
Yeast Complementation Assay for Ion Sensing
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Yeast cells were cultured from −80 °C stock as above. Complementation assays were performed in clear 96-well plates (well capacity: 400 µl). Each well contained 200 µl yeast culture. Medium was made from 100 µl 2x SG medium and 80 µl of CaCl2, BAPTA or MnSO4 (all dissolved in 100 mM TES/KOH, pH = 6) to the desired final concentrations. 20 µl of a cell suspension with OD450 = 0.5 in dH2O was added to each well. Plates were incubated at the desired temperature and OD450 of the cultures measured in a plate-reader (Multiskan RC, Thermo Labsystems) at appropriate time intervals. BAPTA (1, 2-bis(o-Aminophenoxy)ethane-N,N,N’,N’-tetraacetic Acid) was purchased as tetrasodium salt from Merck (cat.no.: 196418).
4
Evaluating Cytotoxicity of Recombinant MeV
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Respective cells of interest (1 × 104) were seeded into 96-well plates (Nunc) and infected with recombinant MeV (MOI = 1) or left uninfected (mock) 4 h after seeding in quadruplicates. Viability of the cells after infection was determined using MTT (Cell Proliferation Kit I; Roche Diagnostics). Cells were incubated with the MTT solution for 4 h and then solubilization solution was added 72 h after infection. Following overnight incubation, a formazan dye was formed, which was quantified in quadruplicates using an ELISA reader (Multiskan RC; Thermo Labsystems, Dreieich, Germany). Averages of the replicates were calculated and values of virus infected samples were divided by those of uninfected controls to calculate relative cytotoxicity in %.
5
Evaluating Osteoblast Responses to Zoledronate
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Cell proliferation was analyzed using XTT assays (Biological Industries, Kibbutz Beit-Haemek, Israel), for which the cells were plated in 96-well plates and allowed to attach for 24 h. The culture media were then changed to the ZOL-conditioned and control media for a 48-h treatment. An ELISA plate reader (Thermo Labsystems Multiskan RC, Vantaa, Finland) was used to measure the absorbance of the samples at a wavelength of 490 nm.
Apoptosis was detected using chromatin condensation and fragmentation with H342 dye (Sigma-Aldrich, St. Louis, MO, USA). Cells were seeded in 6-well plates, grown for 24 h, and then incubated with the ZOL-conditioned and control media for 48 and 72 h. H342 dye from the stock solution, originally suspended in 1 mM of ddH2O, was added to the cell suspension for a total of 10 μM. The cells were then incubated for 60 min, and the apoptotic cells were examined under a fluorescence microscope.
We studied the effects of ZOL at the μM level on osteoblast recruitment using a cell migration assay. Cells were seeded in 6-well plates, allowed to attach for 24 h, and then incubated with the ZOL-conditioned and control media for 48 h. After they had been treated, a pipette tip was used to make an I-shaped scratch on the well. The scratch was followed for its closure and photographs were taken under a microscope every 12 h for 3 days.
Apoptosis was detected using chromatin condensation and fragmentation with H342 dye (Sigma-Aldrich, St. Louis, MO, USA). Cells were seeded in 6-well plates, grown for 24 h, and then incubated with the ZOL-conditioned and control media for 48 and 72 h. H342 dye from the stock solution, originally suspended in 1 mM of ddH2O, was added to the cell suspension for a total of 10 μM. The cells were then incubated for 60 min, and the apoptotic cells were examined under a fluorescence microscope.
We studied the effects of ZOL at the μM level on osteoblast recruitment using a cell migration assay. Cells were seeded in 6-well plates, allowed to attach for 24 h, and then incubated with the ZOL-conditioned and control media for 48 h. After they had been treated, a pipette tip was used to make an I-shaped scratch on the well. The scratch was followed for its closure and photographs were taken under a microscope every 12 h for 3 days.
6
Quantification of VEGF and TGF-β
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Measurements of intracellular and serum levels of VEGF and transforming growth factor β (TGF-β) were performed using Quantikine ELISA kits (VEGF: MMV00, TGF-β: DB100B, R&D systems, USA) according to the manufacturer’s instructions using 500 µg of brain protein and fivefold diluted serum. Optical density was measured at 450 nm, with wavelength correction at 570 nm (Multiskan RC; Thermo Labsystems, Finland).
7
Cytokine Quantification using ELISA
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Cytokine determination in the preserved supernatants was performed using the ELISA kit, TNF-α and IL-6, according to the attached protocol. Cytokine levels in the supernatants were determined using Biolegend ELISA kits for TNF-α and IL-6 according to the manufacturer’s protocols. The optical density was measured using a Multiskan RC spectrophotometric reader (Thermo Labsystems, Waltham, MA, USA) at λ = 450 nm.
8
Evaluating Cell Membrane Integrity
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25 000 cells per well (in a volume of 150 μL) were seeded in 96-well-plates and were allowed to adhere overnight. Cells were exposed to BAL-like solutions (neutralized or not) and after a 24 h incubation cell membrane integrity was evaluated. The cellular release in the supernatant of cytoplasmic lactate dehydrogenase (LDH) was assessed using the CytoTox-96™ Homogeneous Membrane Integrity Assay (Promega) according to the manufacturer's instructions. The optical density of the samples was determined using a microplate reader (Multiskan RC, Thermolabsystems) set to 490 nm. Three independent experiments were performed, each in quadruplicate and the activity of the released LDH was reported to that of negative control cells (cells alone). A positive control consisting of the maximal cellular LDH released after cells lysis was used.
To determine potential colorimetric bias, experiments were performed in different media: either DMEM containing phenol red (Sigma-Aldrich) or DMEM without phenol red (Pan-Biotech).
To determine potential colorimetric bias, experiments were performed in different media: either DMEM containing phenol red (Sigma-Aldrich) or DMEM without phenol red (Pan-Biotech).
9
MTT Assay for Cell Viability
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A MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to evaluate cell viability. Briefly, 15 000 cells per well (in a volume of 150 μL) were seeded in 96-well-plates and were allowed to adhere overnight. Cells were exposed to BAL-like solutions (neutralized or not) and after a 24 h incubation 20 μL of MTT (5 μg mL−1, Sigma-Aldrich) were added in each well, and plates were incubated for 3 h at 37 °C. Then, 200 μL of dimethylsulfoxide (DMSO, ThermoFisher Scientific) was added and thoroughly mixed to dissolve the crystals. The optical density of each sample was determined using a microplate reader (Multiskan RC, Thermolabsystems) set to 570 nm. Negative and positive controls were included: cells incubated alone and cells incubated in the presence of 0.1% Triton X-100 (Sigma-Aldrich).
For the bias study, cell culture supernatant was removed by aspiration before the addition of 200 μL of fresh culture medium and 20 μL of MTT. The other steps of the protocol remained unchanged.
For the bias study, cell culture supernatant was removed by aspiration before the addition of 200 μL of fresh culture medium and 20 μL of MTT. The other steps of the protocol remained unchanged.
10
Viability Assay of Breast Cancer Cells
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The viability of MDA-MB-231 and BT-549 cells (5.0 × 103 cells/well, 96-well plates), untreated and treated with ψRGDechi (from 1 to 50 µM), at times of 24, 48, and 72 h was assessed as previously reported [48 (link)] by CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega BioSciences Inc., Fitchburg, WI, USA) using 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS), and according to the manufacturer’s instructions. After 1-hour incubation with MTS, absorbance was read in a plate reader (Multiskan RC, Thermo Scientific, Waltham, MA, USA) at a wavelength of 490 nm. The data are expressed as percentage of viable cells, considering the untreated control cells as 100%.
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