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Live dead solution

Manufactured by Thermo Fisher Scientific
Sourced in United States

The Live/Dead solution is a laboratory reagent used to assess cell viability. It contains fluorescent dyes that can distinguish between live and dead cells. The solution is designed to provide a quick and reliable method for evaluating the health and integrity of cell populations in various applications.

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5 protocols using live dead solution

1

Cell Viability Quantification in Agarose Constructs

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Cell-agarose constructs were divided into two halves, washed with 1× PBS and incubated in 700 μL live/dead solution (Thermo Fisher Scientific, Cat: L3224) for 18 min to stain live and dead cells using green fluorescent calcein-AM (4 mM) and red fluorescent ethidium-homodimer-1 (2 mM), respectively. Then, samples were washed with 1× PBS to remove the residual dye and imaged under fluorescence imaging using a Nikon TiE microscope with a DS-Qi2 camera (Nikon Instruments Inc., Melville, NY, USA). 10× and 4× cross-sectional images were taken of each gel with 10× images used for automatized quantification of cell viability (percentage live cells =s number of live cells over total cells) using MIPAR Image Analysis Software (Tang et al., 2019 (link)).
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2

Cell Viability and Localization in Scaffolds

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Cell-laden printed scaffolds (n = 3 per group) were washed with PBS and then immersed in the LIVE/DEAD solution (ThermoFisher, Waltham, MA, USA) for 15 min at 37 °C after 3 and 7 days of culture. After 15 min, the LIVE/DEAD solution was removed, and the samples were washed with PBS and stained with DAPI (Sigma, St. Louis, MO, USA) for 15 min at 37 °C. Scaffolds were washed with PBS once more and then visualized using a fluorescent microscope (Zeiss Axiovert) (Zeiss, Dublin, CA, USA) with Lumenera Infinity 3 (Teledyne, ON, Canada). Cell viability measurements were determined through ImageJ. The presence of cells within the cell-laden scaffolds were also confirmed with DAPI. Briefly, cell-laden scaffolds (n = 3 per group) were fixed with 10% formalin (Sigma, St. Louis, MO, USA) for 15 min and then washed with PBS. Afterwards, the scaffolds were placed into Tissue-Tek (Leica, Wetzlar, Germany) at −80 °C for 30 min and then sectioned at 20 µm using a cryomicrotome (Leica, Wetzlar, Germany). The cryosections were then stained with DAPI for 5 min at 37 °C and visualized with the fluorescent microscope.
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3

Cell Viability Quantification in Agarose Constructs

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Cell-agarose constructs were divided into two halves, washed with 1× PBS and incubated in 700 μL live/dead solution (Thermo Fisher Scientific, Cat: L3224) for 18 min to stain live and dead cells using green fluorescent calcein-AM (4 mM) and red fluorescent ethidium-homodimer-1 (2 mM), respectively. Then, samples were washed with 1× PBS to remove the residual dye and imaged under fluorescence imaging using a Nikon TiE microscope with a DS-Qi2 camera (Nikon Instruments Inc., Melville, NY, USA). 10× and 4× cross-sectional images were taken of each gel with 10× images used for automatized quantification of cell viability (percentage live cells =s number of live cells over total cells) using MIPAR Image Analysis Software (Tang et al., 2019 (link)).
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4

Evaluating Cell Viability via Live/Dead Staining

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Cytotoxicity was evaluated by staining cells with a live/dead solution (Invitrogen) according to the manufacturer’s instructions. Cells stained with green fluorescence for live cells and red fluorescence for dead cells were observed using a confocal microscope (LSM 880, Carl Zeiss, Oberkochen, Baden-Württemberg, Germany). The percentage of viable cells was calculated by counting the live and dead cells in the confocal images.
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5

Phenotypic Analysis of PBMC Subsets

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Antibodies used to detect cell surface markers of PBMCs were: anti-human CD3-Alexa Fluor 700, anti-human CD4-FITC, anti-human CD25-APC-eFluor 780, anti-human CD39-PE-Cy7 and anti-human CD56-PE from eBioscience; anti-human CD8-Pacific Blue, anti-human CXCR3 PE-Cy5 and PE-Cy5 mouse IgG1, κ isotype control from BD Bioscience.
Cells were resuspended in 5 μl Live/Dead solution (Invitrogen-Life Technologies) for 10 min at RT, then incubated with cell surface antibodies for 30 min at RT followed by intracellular staining for Foxp3 using the anti-mouse/rat/human FOXP3 (Biolegend) following the manufacturer's instructions. Flow cytometry analysis was performed using a LSRII (BD Bioscience) and data analyzed with FlowJo software.
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