Single‐Cell and BioMark HD Systems (Fluidigm, San Francisco, CA, USA) were used for specific target amplification in NPCs, neurons, and organoids as described (Chailangkarn et al, 2016 ; Trujillo et al, 2019 ). Briefly, single cells were captured on a C1 chip, and a LIVE/DEAD Cell Viability/Cytotoxicity kit (Life Technologies) was used to assess viability. DELTAgene primer pairs (96.96 Dynamic Array IFC chip) were used for single‐cell qPCR; results analysis was performed using Fluidigm Real‐time PCR Analysis Software and Singular Analysis Toolset 3.0. The full list of markers used can be found in Dataset EV1 .
Live dead cell viability cytotoxicity kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The Live/Dead cell viability/cytotoxicity kit is a fluorescence-based assay used to determine the percentage of live and dead cells in a cell population. The kit uses two fluorescent dyes to distinguish between live and dead cells. One dye stains only dead cells, while the other dye stains all cells. The kit provides a quantitative measure of cell viability and cytotoxicity.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Lsm 710, by Zeiss (4 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Axio observer z1, by Zeiss (3 mentions)
Biomark hd system, by Standard BioTools (2 mentions)
Methacrylic anhydride, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Glutaraldehyde in 0.1 m sodium cacodylate buffer solution, by Electron Microscopy Sciences (2 mentions)
Wst 1, by Merck Group (2 mentions)
Singular analysis toolset 3, by Standard BioTools (1 mentions)
Real time pcr analysis software, by Standard BioTools (1 mentions)
Fibronectin, by Yeasen (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Solarbio (1 mentions)
2 4 6 trinitrobenzenesulfonic acid tnbs, by Merck Group (1 mentions)
24 protocols using live dead cell viability cytotoxicity kit
1
Single-Cell Transcriptomics of NPCs, Neurons, and Organoids
2
Bovine Serum Albumin-Methacrylic Anhydride Hydrogel
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Bovine serum albumin (BSA) and methacrylic anhydride (MAA) were ordered from Sigma-Aldrich (Shanghai, China). Ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) both were purchased from Rhawn (Shanghai, China). Cell counting Kit-8 (CCK-8) and lactate dehydrogenase-cytotoxicity assay kit (LDH) were purchased from Dojindo Molecular Technologies (Fuzhou, China). Collagen type I was purchased from Macklin (Shanghai, China), and fibronectin was purchased from Yeasen (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotic/antimycotic were purchased from Gibco. Live/Dead® Cell Viability/Cytotoxicity kit was purchased from Life Technologies (Shanghai, China). Fialuridine (FIAU) was ordered from Tokyo Chemical Industry (Tokyo, Japan).
3
Rabbit BMSC Viability and Proliferation on Scaffold
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Rabbit bone marrow mesenchymal stem cells (BMSCs)
were cultured in a humidified incubator at 37 °C with 5% CO2. The cells were incubated in a complete medium containing
89% Dulbecco’s modified Eagle medium (DMEM, HyClone), 10% fetal
bovine serum (FBS, Gibco), and 1% penicillin/streptomycin (Solarbio).
The culture medium was changed every two days. Only three-passage
BMSCs were used in the experimental studies.
One milliliter
of the cell suspension (2 × 105/mL) was dropped onto
the scaffold and cocultured at 37 °C with 5% CO2.
After culturing for 1, 3, and 5 days, the viability of the BMSCs was
examined by staining the samples using a LIVE/DEAD cell viability/cytotoxicity
kit (Life Technologies) following the manufacturer’s instructions.
Images were captured using a confocal laser scanning microscope (LSM710,
Carl Zeiss, Germany). Living cells showed green fluorescence, whereas
dead cells showed red fluorescence. The proliferation of BMSCs cultured
on the scaffold was detected using a cell counting kit-8 (CCK-8) assay.
To visualize the morphology of BMSCs on the scaffold, TRITC phalloidin
and 4′,6-diamidino-2-phenylindole (DAPI) were used to stain
the cells, and images were taken using a confocal laser scanning microscope;
the cytoskeleton showed red fluorescence, while the nucleus showed
blue fluorescence.
were cultured in a humidified incubator at 37 °C with 5% CO2. The cells were incubated in a complete medium containing
89% Dulbecco’s modified Eagle medium (DMEM, HyClone), 10% fetal
bovine serum (FBS, Gibco), and 1% penicillin/streptomycin (Solarbio).
The culture medium was changed every two days. Only three-passage
BMSCs were used in the experimental studies.
One milliliter
of the cell suspension (2 × 105/mL) was dropped onto
the scaffold and cocultured at 37 °C with 5% CO2.
After culturing for 1, 3, and 5 days, the viability of the BMSCs was
examined by staining the samples using a LIVE/DEAD cell viability/cytotoxicity
kit (Life Technologies) following the manufacturer’s instructions.
Images were captured using a confocal laser scanning microscope (LSM710,
Carl Zeiss, Germany). Living cells showed green fluorescence, whereas
dead cells showed red fluorescence. The proliferation of BMSCs cultured
on the scaffold was detected using a cell counting kit-8 (CCK-8) assay.
To visualize the morphology of BMSCs on the scaffold, TRITC phalloidin
and 4′,6-diamidino-2-phenylindole (DAPI) were used to stain
the cells, and images were taken using a confocal laser scanning microscope;
the cytoskeleton showed red fluorescence, while the nucleus showed
blue fluorescence.
4
Viability of BMSCs on Hydrogel
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The hydrogel sample was prepared and sterilized following the same method as described in Section 4.6.1 . A total 1 ml BMSCs suspension with cell concentration of 2 × 105/mL was dropped onto the surface of the hydrogel and cultivated at 37 °C with 5% CO2. After cultivation for 1 and 3 days, the BMSCs on the surface of the hydrogel were stained by Live/Dead Cell Viability/Cytotoxicity Kit (Life Technologies). Images of the stained BMSCs were observed by confocal laser scanning microscope (LSM710, Carl Zeiss, Germany). Living BMSCs showed green fluorescence, whereas dead BMSCs showed red fluorescence [12 (link)].
5
Gelatin-based Biomaterial Synthesis
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Gelatin (type B, 250 bloom), sodium carbonate, sodium bicarbonate decahydrate, sodium hydroxide, acetohydroxamic acid, hydroxylamine, sodium dodecyl sulfate, and alanine were purchased from Aladdin (Shanghai, China). Methacrylic anhydride (MAA), iron(III) perchloride, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), collagenase (IA, 125 CDU/mg), and 2,4,6-trinitrobenzene sulfonic acid (TNBS) were purchased from Sigma-Aldrich (Shanghai, China). Deuterium oxide (D2O) and 2,2,3,3-D4 (D, 98%) sodium-3-trimethylsilylpropionate (TMSP) were obtained from Cambridge Isotope Laboratories (Andover, USA). Hanks’ Balanced Salt Solution (HBSS) (10X), Dulbecco’s Modified Eagle’s Medium, fetal bovine serum, penicillin/streptomycin, and Live/Dead® Cell Viability/Cytotoxicity kit were purchased from Life Technologies (Shanghai, China). All the reagents were used as received.
6
Evaluating Cell Viability with SSA Treatment
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The effect of SSA on cell viability was analyzed by using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). HUVECs, 4T1 or HCT-15 cells, were seeded at a density of 6 × 103 cells/well in 96-well plates (Corning, United States). After 24-h incubation, the cells were treated with various concentrations of SSA (1–100 μM) for 48 h. Then 10 μL CCK-8 solution was added for an additional 2-h incubation at 37°C. All experiments were performed in triplicate. Absorbance at 450 nm was measured using a microplate reader. Cell viability (%) was calculated against the control.
The LIVE/DEAD cell viability/cytotoxicity kit (Life technology, Carlsbad, CA) was also used to assess the viability of HUVECs. Calcein-AM can be transformed into calcein with green fluorescence in live cells, and propidium iodide (PI, red fluorescence) can stain the nuclei of dead cells. HUVECs (6 × 103 cells/well) were cultured in 96-well plates. After incubation at 37°C for 12 h, the medium was replaced by 200 μL of 1–100-μM SSA for 48-h incubation. Then the culture medium was replaced with 1 ml PBS containing 2 μM calcein-AM (Ex 488 nm and Em 515 nm) and 4.5μM PI (Ex 535 nm, Em 615 nm) for 15 min to stain live and dead cells.
The LIVE/DEAD cell viability/cytotoxicity kit (Life technology, Carlsbad, CA) was also used to assess the viability of HUVECs. Calcein-AM can be transformed into calcein with green fluorescence in live cells, and propidium iodide (PI, red fluorescence) can stain the nuclei of dead cells. HUVECs (6 × 103 cells/well) were cultured in 96-well plates. After incubation at 37°C for 12 h, the medium was replaced by 200 μL of 1–100-μM SSA for 48-h incubation. Then the culture medium was replaced with 1 ml PBS containing 2 μM calcein-AM (Ex 488 nm and Em 515 nm) and 4.5μM PI (Ex 535 nm, Em 615 nm) for 15 min to stain live and dead cells.
7
Hyaluronic Acid Hydrogel for Cell Culture
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The activating reagents for amide bond formation, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were supplied by Acros Organics (Geel, Belgium). N-isopropylacrylamide (NIPAM) and 2,2′-azobis(isobutyronitrile) (AIBN) obtained from Showa Chemical (Tokyo, Japan) were recrystallized from n-hexane and methanol, respectively. Hyaluronic acid (HA) (average molecular weight = 1.3 × 106 Da) was purchased from Bloomage Freda Biopharm Co. (Jinan, China). Alcian blue, Safranin O, 2-morpholinoethane sulfonic acid (MES), 2-aminoethanethiol (AET), 2,4,6-trinitrobenzene sulfonic acid (TNBSA), and Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM-F12) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serums (FBS) and Live/Dead cell viability/cytotoxicity kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). For methyl tetrazolium salt (MTS) assays, the CellTiter96 AQueous One Solution Cell Proliferation Assay kit was obtained from Promega (Madison, WI, USA).
8
Methacrylic Anhydride-Crosslinked Hydrogel
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Methacrylic anhydride (MAA, 94%), Bovine serum albumin (BSA), Deuterium oxide (D2O), Tris(hydroxymethyl)aminomethane, Proteinase K, Dulbecco’s phosphate-buffered saline (DPBS), and paraformaldehyde were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). N, N, N′, N′-tetramethylethylenediamine (TEMED, ≥99.5%) and Ammonium persulfate (APS, >98%) have been bought by Shanghai yi en chemical technology Co., Ltd. (Shanghai, China). Human Skin Fibroblast (HSF) cells were purchased from Kunming Cell Bank, China. Trypsin-EDTA, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S) were purchased from Gibco, Life Technologies, Beijing, China. Live/Dead Cell Viability/Cytotoxicity kit, and Rhodamine phalloidin (R415) were provided from Thermo Fisher Scientific Inc (Waltham, MA, USA). CCK8 kit was purchased by Dojindo Molecular Technologies (Fuzhou, China).
9
Quantifying Cell Viability by Live/Dead Assay
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To quantify cell viability, live and dead cells were stained using a LIVE/DEAD Cell Viability/Cytotoxicity Kit (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol. Briefly, calcein AM was used to stain live cells and ethidium homodimer-1 was used to stain dead cells. Cells were exposed a mixture of the two dyes and incubated at room temperature for 15 min. Cells were counted using live images randomly captured by fluorescence microscopy, and analyzed using Image J software.
10
Islet Viability Evaluation with Live/Dead Assay
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The viability of freshly isolated islets as well as of the islets cultured in control and experimental groups was evaluated on day 1, 4 and 7 using the LIVE/DEAD® Cell Viability/Cytotoxicity Kit (Thermo Fisher Scientific, MA, USA). Islets alone or with scaffolds were placed in a Petri dish, mixed with the prepared staining solution at a 2:1 proportion and dark-incubated for 15–30 min. The result was evaluated using a luminescent microscope (Nikon TS-100, Tokyo, Japan).
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