3D melanoma spheroids were prepared using the liquid overlay method as described in (23 (link)) before being treated with vehicle (dimethyl sulfoxide) or 300nM – 3µM of BI-847325 (Boehringer Ingelheim) for 48h. Spheroids were washed, stained with Live/Dead viability stain (Invitrogen/ Life Technologies Corp.) and analyzed as described in (14 ). The percentage of dead cells was determined using ImageJ software.
Live dead viability stain
Manufactured by Thermo Fisher Scientific
The Live/Dead viability stain is a fluorescent dye used to assess cell viability in a sample. It distinguishes between live (intact) and dead (compromised) cells by staining the latter. This stain is commonly used in various cell-based assays and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Sp5 upright confocal microscope, by Leica (2 mentions)
Bi 847325, by Boehringer Ingelheim (1 mentions)
300 inverted fluorescence microscope, by Nikon (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Evos fl cell imaging system, by Thermo Fisher Scientific (1 mentions)
Fixation buffer, by BD (1 mentions)
Attune acoustic focusing cytometer, by Thermo Fisher Scientific (1 mentions)
Perm buffer 3, by BD (1 mentions)
8 protocols using live dead viability stain
1
Assessing Drug Effects on 3D Melanoma Spheroids
2
Single-Cell Genomic DNA Preparation
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Freely cultured cells were singulated and washed 5 times with C1 DNA Seq Cell Wash Buffer. Cells were counted and loaded onto C1 Single-Cell Open App IFC (Fluidigm cat. no. 100–8133) according to the manufacturer’s instructions. LIVE/DEAD® viability stain (Invitrogen) was included to enable imaging of the capture sites after cell loading and capture. All capture sites were imaged using a Leica® microscope where phase contrast and fluorescent images with GFP and Y3 filters were acquired to determine the number of cells captured, as well as the viability of each of the captured cells. The cells then were lysed and genomic DNA was fragmented with Mu transposase at the same enzyme to DNA ratio as in the tube tagmentation reaction and amplified using the MuEnd primer by keeping the same concentrations of primer and master mix as in the tubes.
3
Uveal Melanoma Spheroid Viability Assay
4
Quantifying Microglia Aβ42 Uptake
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Amyloid Beta 1-42 peptide (Aβ42) conjugated to HiLyte 488 (Anaspec, Fremont, CA) was initially resuspended and fibrilized as reported in16 (link),17 (link). Briefly, Aβ42 was resuspend in 10 nM NaOH (10% of final volume) and then the concentration was adjusted to 1 mg/ml using PBS. Resuspended Aβ42 was frozen at − 20 °C until use. Aβ42 was thawed and incubated at 37 °C overnight (12–14 h) to fibrilize prior to the phagocytosis assay. Aβ42 was added to culture with either young or aged enriched microglia. Duration of culture is noted in each figure. After microglial enrichment and/or phagocytosis assay, samples were stained for flow cytometry using a fixable LiveDead viability stain (ThermoFisher Scientific) and surface stained with the following antibodies: anti-CD45 (clone: 104), -CD11b (clone: M1/70) (Biolegend), and -TREM2 (clone: 237920) (R&D Systems). Aβ42 uptake was evidenced by the presence of HiLyte 488 in microglia (CD45 intermediate and CD11b+ cells) and was quantified via flow cytometry. Data was acquired on a FACSCanto flow cytometer from BD Biosciences and analyzed using FlowJo software (Ashland, OR) or an imaging flow cytometer (Amnis ImageStreamX Mark II, Luminex).
5
Single-Cell mRNA Sequencing of Viable Cells
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Cells were processed and sequencing libraries were prepared according to the Fluidigm C1 Single-Cell mRNA Seq HT IFC v2 protocol, using a medium (10–17μm) integrated fluidic circuit (IFC). For quality control, samples were stained with a LIVE/DEAD viability stain (ThermoFisher) and each capture site was visually inspected using an EVOS FL Cell Imaging System (ThermoFisher) to score the number and viability of cells in each capture site. Successful library tagmentation was assessed using the Advanced Analytical Fragment Analyzer and sample molarity was calculated using the average fragment size. Samples were pooled at equal molarity, spiked with 20% PhiX, and sequenced together in a single high-output 150 cycle NextSeq500 run with read 1 set to 26bp and read 2 set to 75bp.
6
Live/Dead Staining of Encapsulated Cells
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NP cells in 2% agarose gels were stained with LIVE/DEAD viability stain (Life Technologies L3224) as per the manufacturer's instructions, and multiple fields of view were imaged using a Leica SP5 upright confocal microscope with dipping lens. Green staining identified viable cells, while nonviable cells were red or had red nuclei with green cytoplasm. Encapsulated chondrocytes have been reported to remain viable in a 3% agarose gel with a thickness of 3 mm cultured for 9 days, with viability unaffected by spatial distribution.31 The agarose constructs in this study had a thickness of 1.5 mm in order to minimize any diffusion‐related effects on viability.
7
Flow Cytometric Analysis of Transcription Factors in Macrophages
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Expression transcription factors in M0 and M1 macrophages treated or not
for 1 h with the supernatant of cervical-derived carcinoma cell lines
HeLa, SiHa, or C-33A were assessed by FC analysis. Briefly, M0 or M1
macrophages in assay tubes were stained with LIVE/DEAD®viability stain (Life Technologies Corp.) for 30 min to discriminate
between viable and non-viable cells. Afterward, cells were washed
first with PBS and then with stain buffer (FBS) (BD Pharmingen, San
Jose, CA, USA), re-suspended by vortexing in 250 µl of Fixation buffer
(BD™ Phosflow) and immediately incubated for 20 min at 4°C. Fixed
cells were washed with stain buffer and immediately permeabilized, by
slowly adding and under vortex conditions, the cold Perm Buffer III
(BD™ Phosflow). Cells were incubated on ice for 30 min and then washed
with 2 ml of stain buffer. Cells were re-suspended in 100 µl of stain
buffer and were stained with the corresponding Abs for 30 min at 4°C.
Afterwards, the incubated cells were washed and re-suspended in stain
buffer for analysis by FC. Attune Acoustic Focusing Cytometer (Life
Technologies, Carlsbad, CA, USA) was utilized to acquire 20,000 events
in the region of viable cells. Data were processed with FlowJo ver.
10.0.8 statistical software (Tree Star, Inc., Ashland, OR, USA).
Results are reported as percentage of expression or as the geometric
Mean Fluorescence Intensity (MFI).
for 1 h with the supernatant of cervical-derived carcinoma cell lines
HeLa, SiHa, or C-33A were assessed by FC analysis. Briefly, M0 or M1
macrophages in assay tubes were stained with LIVE/DEAD®viability stain (Life Technologies Corp.) for 30 min to discriminate
between viable and non-viable cells. Afterward, cells were washed
first with PBS and then with stain buffer (FBS) (BD Pharmingen, San
Jose, CA, USA), re-suspended by vortexing in 250 µl of Fixation buffer
(BD™ Phosflow) and immediately incubated for 20 min at 4°C. Fixed
cells were washed with stain buffer and immediately permeabilized, by
slowly adding and under vortex conditions, the cold Perm Buffer III
(BD™ Phosflow). Cells were incubated on ice for 30 min and then washed
with 2 ml of stain buffer. Cells were re-suspended in 100 µl of stain
buffer and were stained with the corresponding Abs for 30 min at 4°C.
Afterwards, the incubated cells were washed and re-suspended in stain
buffer for analysis by FC. Attune Acoustic Focusing Cytometer (Life
Technologies, Carlsbad, CA, USA) was utilized to acquire 20,000 events
in the region of viable cells. Data were processed with FlowJo ver.
10.0.8 statistical software (Tree Star, Inc., Ashland, OR, USA).
Results are reported as percentage of expression or as the geometric
Mean Fluorescence Intensity (MFI).
8
Live/Dead Cell Viability Imaging
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NP cells in 6-well culture plates were stained with LIVE/DEAD® viability stain (Life Technologies L3224) as per the manufacturer’s instructions and imaged using a Leica SP5 upright confocal microscope with dipping lens. Green staining identified viable cells, while non-viable cells were red, or had red nuclei with green cytoplasm.
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