DC were cultured as described above. After 24 hours of adherence, RPMI 1640 complete medium was replaced with fresh RPMI 1640 complete medium or RPMI metabolite-depleted medium for 3 hours (i.e., RPMI without glucose or glutamine or RPMI without both). Next, the cells were treated as above in complete or depleted media, followed by infection for 17 hours. The cells were labeled with Calcein-AM (2 μM) and EthD-1 (4 μM) and incubated for 20 minutes at room temperature. Cell viability was measured using a Live/Dead Viability/Cytotoxicity assay kit for mammalian cells (Thermo Scientific) with a fluorescent plate reader at 494/517nm and 528/617nm for Calcein-AM and EthD, respectively.
Live dead viability cytotoxicity assay kit for mammalian cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LIVE/DEAD Viability/Cytotoxicity Assay Kit for Mammalian Cells is a fluorescence-based assay designed to determine the viability and cytotoxicity of mammalian cells. The kit utilizes two fluorescent dyes: one that stains live cells and another that stains dead cells. This allows for the simultaneous detection and quantification of both live and dead cells in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (4 mentions)
Bx61 microscope, by Olympus (2 mentions)
Confocal tcs sp5 upright, by Leica (2 mentions)
P aeruginosa, by Merck Group (1 mentions)
Alamarblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Mueller hinton broth (mhb), by Merck Group (1 mentions)
Ultrapure water, by Merck Group (1 mentions)
S aureus, by Merck Group (1 mentions)
Live dead baclight kit, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 microsystems, by Leica (1 mentions)
Dmi8 inverted microscope, by Leica (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Floid microscope, by Thermo Fisher Scientific (1 mentions)
Evos fl, by Thermo Fisher Scientific (1 mentions)
12 protocols using live dead viability cytotoxicity assay kit for mammalian cells
1
Metabolic Modulation of Cell Viability
2
Nano-Delivery of Antimicrobial Zein and Oregano
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Zein, a protein extracted
from maize, was obtained from Flo Chemical Corporation (MA, USA) and
used for the NCs preparation. Oregano EO from Thymbra capitata (100% pure) was kindly provided by the TELIC S.A. (Barcelona, Spain).
6-Deoxy-6-(ω-aminoethyl) AC was purchased from the Centre of
Excellence of Polysaccharide Research (Germany). MHB, obtained from
Sigma-Aldrich (Spain), was used as the growth medium in all antibacterial
tests. Baird-Parker and Cetrimide selective agars for culturing and
enumeration of S. aureus and P. aeruginosa, respectively, were also purchased from Sigma-Aldrich (Spain). The
bacterial strains S. aureus (ATCC 25923), P. aeruginosa (ATCC 10145), and the human foreskin fibroblast
cells (ATCC CRL-4001, BJ-5ta) were obtained from American Type Culture
Collection (ATCC LGC Standards, Spain). AlamarBlue cell viability
reagent and Live/Dead BacLight kit (Molecular Probes L7012) were purchased
from Invitrogen, Life Technologies Corporation (Spain). Live/Dead
viability/cytotoxicity assay kit for mammalian cells was obtained
from Thermo Fisher Scientific (Spain). Ultrapure water (Milli-Q plus
system, Millipore) with 18.2 MΩ.cm resistivity was used in all
experiments. All other reagents were purchased from Sigma-Aldrich,
if not specified otherwise.
from maize, was obtained from Flo Chemical Corporation (MA, USA) and
used for the NCs preparation. Oregano EO from Thymbra capitata (100% pure) was kindly provided by the TELIC S.A. (Barcelona, Spain).
6-Deoxy-6-(ω-aminoethyl) AC was purchased from the Centre of
Excellence of Polysaccharide Research (Germany). MHB, obtained from
Sigma-Aldrich (Spain), was used as the growth medium in all antibacterial
tests. Baird-Parker and Cetrimide selective agars for culturing and
enumeration of S. aureus and P. aeruginosa, respectively, were also purchased from Sigma-Aldrich (Spain). The
bacterial strains S. aureus (ATCC 25923), P. aeruginosa (ATCC 10145), and the human foreskin fibroblast
cells (ATCC CRL-4001, BJ-5ta) were obtained from American Type Culture
Collection (ATCC LGC Standards, Spain). AlamarBlue cell viability
reagent and Live/Dead BacLight kit (Molecular Probes L7012) were purchased
from Invitrogen, Life Technologies Corporation (Spain). Live/Dead
viability/cytotoxicity assay kit for mammalian cells was obtained
from Thermo Fisher Scientific (Spain). Ultrapure water (Milli-Q plus
system, Millipore) with 18.2 MΩ.cm resistivity was used in all
experiments. All other reagents were purchased from Sigma-Aldrich,
if not specified otherwise.
3
Cell Viability Assay for Nanoparticle Toxicity
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The assessment of cell toxicity was achieved using Invitrogen's LIVE/DEAD® Viability/Cytotoxicity Assay Kit for mammalian cells (#L3224, ThermoFisher Scientific). Briefly, the cells were plated at a density of 0.5 × 106 cells per 35 mm Ibidi chamber and left to adhere overnight. The cells were exposed to PEG5000-AuNPs and ERα-AuNPs (60 pM) nanotags for 48 h at 37 °C. Prior to the assay, the cells were washed three times with PBS. A solution of Calcein AM (10 μL, 4 mM) and EthD-1 (20 μL, 2 mM) were diluted in 10 mL of PBS. The fluorescent staining solution (750 μL) was then added to the cells at 37 °C for 15 min before removing for imaging using a Leica Microsystems TCS SP8 with continuous wave visible lasers and a Leica DMi8 inverted microscope and DFC 7000T and TL LED cameras. The Leica Application Suite X V.3.1.5.16308 software was used to carry out live/dead studies using a Leica 63× magnification HC PL APO water objective with a 1.2 NA. Intensity and area of fluorescence was measured using Image J (National Institute of Health (NIH)) software with Fiji plug-in to measure the area of fluorescent stain.60 (link)
4
Cell Viability and Cytotoxicity Assays
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The MTS assay was used to measure cell viability and proliferation by using the CellTiter 96® AQueous One Solution Cell Proliferation assay (Promega, Alcobendas, Spain), according to the manufacturer’s protocol. 50 μL of MTS were added in each sample cultured with 250 μL of supplemented medium and incubated for 3 h. A scanning multi-well spectrophotometer was used to measure the absorbance at 490 nm.
The LIVE/DEAD Viability/Cytotoxicity Assay Kit for Mammalian Cells (Invitrogen, Carlsbad, CA, USA) was performed to characterize cell viability, attachment, and distribution. The LIVE/DEAD assay was performed by adding 300 μL of a solution at 4 μM EthD-1 and 2 μM of calcein AM in PBS per sample. Materials were incubated for 30–45 min at room temperature. Then, cells were observed with an Olympus BX61 microscope. Micrographs were taken and processed with Fiji software. Live and dead cells were simultaneously stained with green fluorescent calcein-AM and red fluorescent ethidium homodimer-1, respectively.
The LIVE/DEAD Viability/Cytotoxicity Assay Kit for Mammalian Cells (Invitrogen, Carlsbad, CA, USA) was performed to characterize cell viability, attachment, and distribution. The LIVE/DEAD assay was performed by adding 300 μL of a solution at 4 μM EthD-1 and 2 μM of calcein AM in PBS per sample. Materials were incubated for 30–45 min at room temperature. Then, cells were observed with an Olympus BX61 microscope. Micrographs were taken and processed with Fiji software. Live and dead cells were simultaneously stained with green fluorescent calcein-AM and red fluorescent ethidium homodimer-1, respectively.
5
Live/Dead Cell Viability Assay
6
Cell Viability and Proliferation Assays
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Cell viability and proliferation were tested by MTS assay, using the CellTiter 96® AQueous One Solution Cell Proliferation assay (Promega, Alcobendas, Spain), according to the manufacturer’s protocol. Fifty μL of MTS were added in each sample cultured with 250 μL of supplemented medium and incubated for 3 h. Later, the absorbance was measured at 490 nm with a scanning multi-well spectrophotometer.
The LIVE/DEAD Viability/Cytotoxicity Assay Kit for Mammalian Cells (Invitrogen, Carlsbad, CA, USA) was performed to characterize cell viability, attachment, and distribution. The test discriminates live cells from dead cells by simultaneously staining with green fluorescent calcein-AM (live cells) and red fluorescent ethidium homodimer-1 (dead cells). The LIVE/DEAD assay was performed by adding 300 μL of a solution at 4 μM EthD-1 and 2 μM of calcein AM in PBS per sample. Samples were incubated for 30–45 min at room temperature. Then the cells were observed with an Olympus BX61 microscope. Micrographs were taken and processed with Fiji software.
The LIVE/DEAD Viability/Cytotoxicity Assay Kit for Mammalian Cells (Invitrogen, Carlsbad, CA, USA) was performed to characterize cell viability, attachment, and distribution. The test discriminates live cells from dead cells by simultaneously staining with green fluorescent calcein-AM (live cells) and red fluorescent ethidium homodimer-1 (dead cells). The LIVE/DEAD assay was performed by adding 300 μL of a solution at 4 μM EthD-1 and 2 μM of calcein AM in PBS per sample. Samples were incubated for 30–45 min at room temperature. Then the cells were observed with an Olympus BX61 microscope. Micrographs were taken and processed with Fiji software.
7
Cell Sheet Viability and Deformation Evaluation
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The viability of cell sheets was examined using the LIVE/DEAD Viability/Cytotoxicity Assay Kit for mammalian cells (Invitrogen) according to the manufacturer’s instructions. The cultured cells or transferred cells were gently washed three times with DPBS. Calcein acetoxymethyl (AM) and ethidium homodimer-1 (EthD-1) were diluted together in DPBS. Diluted calcein AM and EthD-1 solution (1 ml) was added to cultured cells and kept for 45 min at room temperature. The live cells were stained with calcein AM, and dead cells were stained with EthD-1. After staining, cells were gently washed with 1× DPBS for three times and imaged with a fluorescence microscope (LSM-880, Carl Zeiss). Off-axis deformation of the cell sheets before and after the delivery process was quantified using SLIM. The optical system was assembled by attaching a SLIM module (CellVista SLIM Pro, Phi Optics) to the output port of an existing inverted phase-contrast microscope (26 (link)).
8
Cell Viability Assay with Kynurenine
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Cells were plated in 6-well plates (250×103 cells/well), cultured and treated with increasing concentrations (50 and 150 μg/ml) of Kyn or KynA. After 3 days of incubation cell viability was evaluated by flow cytometry using the Live/Dead, Viability/Cytotoxicity assay kit for mammalian cells (Invitrogen). In this assay ethidium homodimer (EthD-1), a red fluorescent nucleic acid dye, stains dead cells and those undergo apoptosis; while, clacein AM is converted to a green fluorescent compound by active intracellular esterase in live cells. Percentage of dead cells plus cells with damaged (porous) cellular membrane was quantitatively evaluated based on the protocol suggested by the manufacturer (Molecular Probes, Invitrogen, Mississauga Canada).
9
Assessing Astrocyte Viability Under Hypoxia
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The live/dead assay is used for assessing cell viability/death and was carried out using a live/dead viability/cytotoxicity assay kit for mammalian cells (Invitrogen Detection Technologies, Thermofisher, UK) according to the manufacturer’s instructions. This method is used for assessing cell viability/death in live cell culture of cells. It uses two components, namely; Calcein AM (CaAM), which is component A and Ethidium homodimer-1 (EthD-1), which is component B. Dead cells are characterized by intense fluorescence at over 600 nm and little fluorescence around 530 nm.
Astrocytes seeded in an 8-well chamber slide were allowed to adhere and grow to confluence for 14 h at 37 °C in a CO2 incubator. Thereafter, cells were placed in a normoxic or severe hypoxic condition for 3 and 6 h. After 3 and 6 h, 100 μL of EthD-1 (1 μL/mL) and CaAM (0.2 μL/mL) were separately prepared in cell media (MEM), added to the cells, and then, incubated at 37ºC for 10 min. Fluorescent images of the cells were taken at excitation/emission(ex/em) wavelength of 495 nm/515 nm for the green filter (live cells) and ex/em wavelength of 495 nm/635 nm for the red filter (dead cells) using an Invitrogen Floid microscope (Fisher Scientific, Sweden). Percentage cell death was calculated to get the number of dead cells.
Astrocytes seeded in an 8-well chamber slide were allowed to adhere and grow to confluence for 14 h at 37 °C in a CO2 incubator. Thereafter, cells were placed in a normoxic or severe hypoxic condition for 3 and 6 h. After 3 and 6 h, 100 μL of EthD-1 (1 μL/mL) and CaAM (0.2 μL/mL) were separately prepared in cell media (MEM), added to the cells, and then, incubated at 37ºC for 10 min. Fluorescent images of the cells were taken at excitation/emission(ex/em) wavelength of 495 nm/515 nm for the green filter (live cells) and ex/em wavelength of 495 nm/635 nm for the red filter (dead cells) using an Invitrogen Floid microscope (Fisher Scientific, Sweden). Percentage cell death was calculated to get the number of dead cells.
10
Cell Proliferation and Viability Assays
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Cell proliferation has been tested using the MTS assay CellTiter 96® AQueous One Solution Cell Proliferation assay (Promega) according to manufacturer's protocol. 50 μl of MTS were added in each sample cultured with 250 μl of supplemented medium, incubating for 3 hours and then recording the absorbance at 490 nm. LIVE/DEAD Viability/Cytotoxicity assay Kit for Mammalian Cells (Invitrogen) was performed in order to characterize cell viability, attachment and distribution. It discriminates live from dead cells by simultaneously staining with green-fluorescent calcein-AM (live cells) and red-fluorescent ethidium homodimer-1 (dead cells). Live/Dead assay was performed by adding 300 μl of a solution at 4 μM EthD-1 and 2 μM of calcein AM in Phosphate-buffered saline (PBS), per sample and incubated for 30-45 min at room temperature. Then, surfaces have been observed with a Confocal TCS SP5 Upright from Leica Microsystems and the images were processed with Fiji software.
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