Cultured primary mixed cortical cells were incubated with nanoparticle variants at defined final nanoparticle concentrations, and stained using the Live/Dead™ reagent (4 μM ethidium homodimer-1 (EthD-1) and 2 μM calcein AM) (Molecular Probes™) to identify live versus dead cells on the basis of membrane integrity and esterase activity. After a 45 minute incubation at room temperature, cytotoxicity was quantified by recording fluorescence with a plate reader set to 494/517 nm (excitation/emission) for calcein AM (live cells) and 528/617 nm for EthD-1 (dead cells). Experiments were repeated three times using cultures derived from separate litters of rat pups. Representative wells were imaged using fluorescence microscopy with a 40× objective lens to assess four standardized fields of view (FOV) of 293.703 × 293.703 μm per technical replicate (n = 3). The numbers of appropriately florescent live and dead cells were counted and expressed as mean percentages of the total cell population within the FOV.
Live dead reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Live/Dead reagent is a fluorescent dye-based solution used to differentiate between live and dead cells in a sample. It provides a rapid and reliable method for assessing cell viability.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd56 pe cy7, by BioLegend (3 mentions)
Anti cd3 pe cf594, by BD (3 mentions)
Cytotoxicity detection kit ldh, by Roche (2 mentions)
Cell trace violet proliferation dye, by Thermo Fisher Scientific (2 mentions)
Confocal laser scanning microscope, by Olympus (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
Syto9, by Thermo Fisher Scientific (2 mentions)
Las af software, by Leica (2 mentions)
Glass coverslip, by World Precision Instruments (2 mentions)
Celltiter glo reagent, by Promega (2 mentions)
Inverted fluorescence microscope, by Olympus (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Fc500 cytometer, by Beckman Coulter (1 mentions)
Propidium iodide, by BD (1 mentions)
Canto 2, by BD (1 mentions)
Celltrace violet cell proliferation dye, by Thermo Fisher Scientific (1 mentions)
Axiovert 1 microscope, by Zeiss (1 mentions)
Lsr fortessa 2, by BD (1 mentions)
Anti cd3 apc efluor780, by Thermo Fisher Scientific (1 mentions)
Facsdiva software, by BD (1 mentions)
36 protocols using live dead reagent
1
Evaluating Nanoparticle Cytotoxicity in Primary Neurons
2
Cytotoxicity Evaluation of Silver Acetate/Plazomicin
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Cytotoxicity was assessed on HEK293 cells [66 (link)]. The methodology followed was described previously [67 (link)]. Cytotoxicity was determined for of the combination silver acetate/plazomicin at various concentrations. One thousand cells per well were cultured on flat-bottom 96-well, black microtiter plates. After 12 h incubation, the testing compounds were added at the desired concentrations and incubation was continued for 24 h. Following, the cells were washed with sterile D-PBS, resuspended in the LIVE/DEAD reagent (2 μM ethidium homodimer 1 and 1 μM calcein-AM) (Molecular Probes), and incubated for 30 min at 37 °C. At this moment the fluorescence levels corresponding to dead and live cells (645 nm and 530 nm, respectively) were measured. The percentage of dead cells was calculated relative to the untreated cells. Maximum toxicity was calculated treating cells with 70% methanol for 20 min. Experiments were conducted in triplicate. The results were expressed as mean ± SD of three independent experiments. HEK293 cells were purchased from BEI resources (Manassas, VA, USA), catalog number NR-9313.
3
Cytotoxicity Evaluation of Silver Acetate/Amikacin
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The cytotoxicity of the formulation silver acetate/amikacin was assessed on HEK293 cells [51 (link)] as described previously [29 (link)]. Briefly, 103 cells per well were inoculated and cultured overnight using flat-bottom 96-well, black microtiter plates. After this period, the compounds to be tested were added to cell-containing wells at the concentrations indicated, and incubation was continued for 24 h. At this point, the cells were washed with sterile D-PBS, resuspended in the LIVE/DEAD reagent (2 μM ethidium homodimer 1 and 1 μM calcein-AM) (Molecular Probes), and incubated for 30 min at 37 °C before determining the fluorescence levels at 645 nm (dead cells) and 530 nm (live cells). The percentage of dead cells was calculated relative to the untreated control cells. The maximum toxicity control was calculated treating the cells with 70% methanol for 10 min. Experiments were conducted in triplicate. The results were expressed as mean ± SD of three independent experiments.
4
Myricetin Protects A549 Cells from S. aureus
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A549 cells were seeded in 96-well-plates at a density of 1.5 × 104 cells per well the day before infection. The S. aureus suspension described above was added to the cells at a multiplicity of infection (MOI) of 50 in the presence of myricetin at the indicated concentrations, and the 96-well-plates were incubated at 37°C for 6, 16, or 24 h. The LDH released into the supernatants was measured with a Cytotoxicity Detection Kit (LDH, Roche) as recommended by the manufacturer. Cells at the bottom of the wells were stained with live/dead reagent (Invitrogen) and observed under an inverted fluorescence microscope (Olympus).
5
Multiparametric Flow Cytometry Analysis of Lymphocytes
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Cells were co-stained with LIVE/DEAD reagent (Invitrogen, Grand Island, NY) and hu-CD3-APC, as well as hu-CD4-PE, hu-CD8-PE, hu-CD19-PE (BD Biosciences, San Diego, CA), or hu-CD56-PE (Beckman Coulter, Brea, CA). Events were gathered by gating on cells negative for the LIVE/DEAD stain on an FC500 cytometer (Beckman-Coulter). Cell viability also was measured by flow cytometry using annexin-V-FITC and propidium iodide (PI) (BD Biosciences). Human HLA-B8 tetramers complexed with immunodominant peptide from BZLF-1 (RAKFKQLL) and conjugated with allophycocyanin (APC) [18 (link)] were provided by the NIAID Tetramer Facility and the NIH AIDS Research and Reference Reagent Program (Atlanta, Georgia). Where indicated, cell counting beads were used to obtain cell numbers according to manufacturer's description (BD Biosciences).
6
Assessing TriKE-Mediated NK Cell Expansion
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To measure the ability of the TriKE to specifically induce NK cell expansion via the IL-15 moiety, PBMCs from healthy donors were labeled with CellTrace Violet Proliferation Dye (Invitrogen, Carlsbad, CA, USA) according to kit specifications. After staining, cells were cultured with TriKEs at noted concentrations, or equimolar concentrations of controls, and incubated in a humidified atmosphere containing 5% CO2 at 37 °C for seven days. Cells were harvested, stained for viability with Live/Dead reagent (Invitrogen, Carlsbad, CA, USA) and surface stained for anti-CD56 PE/Cy7 (Biolegend, San Diego, CA, USA) and anti-CD3 PE-CF594 (BD Biosciences, Franklin Lakes, NJ, USA) to gate on the viable CD56+ CD3- NK cell population or the CD56-CD3+ T cell population. Data analysis was performed using FlowJo software (FlowJo LCC, version 7.6.5, Ashland, OR, USA).
7
Cytotoxicity Assay of Listeriolysin O
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Purified LLO (0.5 μM) was incubated with different concentrations of CT extract for 30 min at 37 °C, and then the mixture was added to the well of 96-well plates to incubate with the cells for 5 h. The cells treated with DMEM and 0.2% Triton X-100 were used as the negative control and the positive control, respectively. The LDH detection method was the same as described previously. In addition, the treated cells were stained with live/dead reagent (Invitrogen, Carlsbad, CA, USA) and then photographed with a confocal laser scanning microscope (Olympus, Tokyo, Japan) [20 (link)].
8
Evaluating S. aureus-Induced Cytotoxicity
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Lung-tissue-related
human lung epithelial cells (A549, ATCC) and mouse alveolar macrophages
(MH-S, ATCC) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM), and approximately 2 × 105 cells per
well were inoculated in a 96-well plate in CO2 incubators
overnight. S. aureus USA300 was grown
in TSB at 37 °C with OA at concentrations of 0–4 μg/mL
until the postexponential growth phase was reached at an OD600 value of 2.5. Bacterial culture supernatants were harvested and
filtered with a 0.22 μm filter. Next, the cells were incubated
with 100 μL of the above culture supernatants of S. aureus USA300 for 6 h at 37 °C. Following
centrifugation (1000 rpm, 10 min), the LDH released in the supernatant
was examined using a lactate dehydrogenase (LDH) test kit (Roche,
Mannheim, Germany). The cells in 96-well plates were treated with
a live/dead reagent (Invitrogen, Carlsbad, CA) to qualitatively evaluate
cell viability. Green fluorescently labeled cells were viable, and
red fluorescently labeled cells were dead.
human lung epithelial cells (A549, ATCC) and mouse alveolar macrophages
(MH-S, ATCC) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM), and approximately 2 × 105 cells per
well were inoculated in a 96-well plate in CO2 incubators
overnight. S. aureus USA300 was grown
in TSB at 37 °C with OA at concentrations of 0–4 μg/mL
until the postexponential growth phase was reached at an OD600 value of 2.5. Bacterial culture supernatants were harvested and
filtered with a 0.22 μm filter. Next, the cells were incubated
with 100 μL of the above culture supernatants of S. aureus USA300 for 6 h at 37 °C. Following
centrifugation (1000 rpm, 10 min), the LDH released in the supernatant
was examined using a lactate dehydrogenase (LDH) test kit (Roche,
Mannheim, Germany). The cells in 96-well plates were treated with
a live/dead reagent (Invitrogen, Carlsbad, CA) to qualitatively evaluate
cell viability. Green fluorescently labeled cells were viable, and
red fluorescently labeled cells were dead.
9
Evaluating TiO2:C Nanoparticle Cytotoxicity in A549 Cells
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A549 cells were seeded in 3.5-cm culture dish at a density of 8.5 × 104 cells/mL and cultured for one day. The synthesized TiO2:C (1 mg/mL) in 100 μL of medium was added to each culture dish. As described in Section 2.3 , cells were irradiated with X-ray generated at 80 kV and 10 mA at a dose rate of 0.08 Gy/min for 100 s. All dishes were incubated at 37 °C in a 5% CO2 atmosphere for one day. In the following three days, l mL of live/dead reagent (1387211, Invitrogen, Carlsbad, CA, USA) prepared with phosphate-buffered saline (PBS) was added into each dish and kept in the dark, in a static state for 30 min. The cells were then examined under a confocal microscope (Olympus, BX51, Tokyo, Japan).
10
Flow Cytometric Analysis of MHC-Peptide Complexes
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Cells were blocked using the block agent from BioLegend, washed with PBS, and stained with Live/Dead reagent (Invitrogen) and antibodies specifically recognizing mouse H-2Kb (Biolegend, Cat#116506) and mouse H2Kb bound to OVA peptide (Biolegend, Cat#141604) for 20 minutes on ice and washed twice. FACS acquisition was done on the BD Canto II using FlowJo software (BD Bioscience) for analysis.
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