pMacpre were seeded at 4 × 104 cells/well in three optically clear bottom black 96-well plates (Costar) and differentiated in macrophage media. After 7 days, a full media change was performed to 100 μL macrophage media ± M-CSF, triplicate wells for each condition on each plate. Plates were incubated at 37 °C/ 5% CO2 for 3, 7, or 10 days, and the 10-day plate received a 50% media change at 7 days. At the end of each incubation, cells were stained 20 min (37 °C/5% CO2) with the ReadyProbes Cell Viability Imaging Kit (Invitrogen). Nuclei counting was performed using an EVOS FL Auto automated microscope (Thermo Fisher), 4x objective with DAPI and GFP light cubes, 4 fields/well, and CellProfiler 2.2 software [33 (link)]. Data was presented as (mean number of dead cells/mean number of total cells) × 100 for each condition.
Readyprobes cell viability imaging kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ReadyProbes Cell Viability Imaging Kit is a fluorescence-based assay for detecting live and dead cells in cell culture samples. It contains two ready-to-use nuclear stains, one for live cells and one for dead cells. The kit provides a simple and convenient method for analyzing cell viability and proliferation in a variety of cell types.
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Lab products found in correlation
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Evos fl microscope, by Thermo Fisher Scientific (2 mentions)
Trypan blue, by Thermo Fisher Scientific (2 mentions)
Lab tek 2 chamber slide system, by Thermo Fisher Scientific (2 mentions)
Evos cell imaging system, by Thermo Fisher Scientific (2 mentions)
Icell8 single cell system, by Takara Bio (2 mentions)
Nucblue live, by Thermo Fisher Scientific (2 mentions)
Nucgreen dead, by Thermo Fisher Scientific (2 mentions)
Evos fl imaging system, by Thermo Fisher Scientific (1 mentions)
Cellcarrier 96 ultra microplate, by PerkinElmer (1 mentions)
Live cell imaging solution, by Thermo Fisher Scientific (1 mentions)
Staurosporine, by MP Biomedicals (1 mentions)
Eclipse ti2 e, by Nikon (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Cellrox deep red reagent, by Thermo Fisher Scientific (1 mentions)
Image it live red poly caspases detection kit, by Thermo Fisher Scientific (1 mentions)
Prism 6, by GraphPad (1 mentions)
Hk 2 cell line, by American Type Culture Collection (1 mentions)
Cell counter, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc cell apoptosis detection kit, by Beyotime (1 mentions)
37 protocols using readyprobes cell viability imaging kit
1
Macrophage Viability Quantification
2
Spheroid Cell Viability Assays
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Cell viability during the spheroid cultures was assessed with the ReadyProbes™ Cell Viability Imaging Kit (Invitrogen, Thermo Fisher Scientific). A drop of each stain, one for cell nuclei and one for dead cells, was added to 500 µL of media with the samples and incubated at +37 °C for 20 min. Samples were then imaged with the EVOS FL imaging system (Thermo Fisher Scientific).
We also used Calcein-AM, which stains live cells green, and ethidium homodimer-1 (EthD), which stains dead cells red by entering cells with plasma membrane damage. Samples were washed twice with PBS and a working solution of 1:8000 of Calcein-AM and EthD was added. Samples were incubated at 37 °C for 30 min in the dark and imaged with a EVOS FL microscope (Thermo Fisher Scientific).
We also used Calcein-AM, which stains live cells green, and ethidium homodimer-1 (EthD), which stains dead cells red by entering cells with plasma membrane damage. Samples were washed twice with PBS and a working solution of 1:8000 of Calcein-AM and EthD was added. Samples were incubated at 37 °C for 30 min in the dark and imaged with a EVOS FL microscope (Thermo Fisher Scientific).
3
Macrophage Survival Assay Protocol
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Macrophage precursors were plated at 4 × 104 cells/well in four separate CellCarrier-96 Ultra Microplates (Perkin Elmer), and differentiated in macrophage medium for 1 week. Survival was assessed as previously described23 (link). In brief, cells received a full media change to fresh macrophage medium with or without M-CSF (100 ng/ml), with triplicate wells per condition on each plate. One plate was used to assess the cell number at baseline (day 0), while the other plates were incubated at 37 °C/ 5% CO2 for a further 3, 7, or 10 days. The 10-day plate received a 50% medium change at day 7. At the end of each incubation, cells were stained with the ReadyProbes Cell Viability Imaging Kit (Invitrogen) for 30 min at 37 °C/5% CO2. Nuclei were imaged using the Opera Phenix High Content Screen System (Perkin Elmer) with a 10 × objective and 9 fields per well. Images were quantified with Columbus 2.7 software (Perkin Elmer). Data was presented as percentage of mean number of dead cells/mean number of total cells for each condition.
4
Cell Viability Imaging with ReadyProbes
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The ReadyProbes™ Cell Viability Imaging Kit (Invitrogen, Thermo Fisher Scientific) was also used to assess cell viability during the chip culture experiments. Two drops of each stain were mixed with 1 mL of medium and the mixture was pipetted into the chips with differing volumes on each side to create a flow through the gel. The chips were incubated 10 min at +37 °C followed by 10 min at RT. The images were taken with an EVOS FL microscope (Thermo Fisher Scientific).
5
Cell Viability Assay with Stimulants
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Viability was determined in unstimulated, 1 ng/mL LPS, 1 µg/mL Pam3CSK4, or 3 µM Staurosporine (MP Biomedicals, #0219140080) stimulated cells. Cells were cultured as per cell titration assay at desired densities and stimulated by 50% media change with 2x agonist containing fresh media. After 24 hours, cells were stained with ReadyProbes™ Cell Viability Imaging Kit (Invitrogen, #R37609) diluted in Live Cell Imaging Solution (Invitrogen, #A14291DJ) for 15 minutes at room temperature. This was then replaced with fresh Live Cell Imaging Solution and cells were immediately visualised on the EVOS® Fl Auto to quantify stained nuclei.
6
Evaluating hMSCs Viability in Macroparticles
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Trypsinized passage 4 hMSCs were centrifuged and resuspended in CM, dextran-based CM or Ficoll-Pq -based CM at a density of 20 0,0 0 0 cells per 37 μl. Cell suspensions were incubated at 37 °C/ 5% CO 2 in plates mimicking the 4h seeding in 3D scaffolds in contact with the macroMs. To assess the viability of cells when in a macroMs solution, incubation was done in presence of NucBlue® Live and NucGreen® Dead reagents (ReadyProbes® Cell Viability Imaging Kit, Invitrogen) as defined by the manufacturer protocol and imaged at t = 0 h and t = 4 h after seeding using an inverted fluorescent microscope (Eclipse, Ti2-e, NIKON). For optimal visualization in the reported images, live cells were false colored in green while dead cells were given a red color. After 4 h, the remaining cell suspensions (without cell viability reagents) were centrifuged, the macroMs containing media was removed and cells were resuspended in fresh CM to mimic the post-seeding culture condition of cells in scaffolds. Cells were counted and seeded at a density of 5,0 0 0 cells cm -2 in tissue culture polystyrene well plates. Media was refreshed every two or three days. To evaluate hMSCs osteogenic differentiation, scaffolds were cultured for 21 days in BM or MM after 7 days in BM.
7
Cell Viability and Apoptosis Assessment
8
Apoptosis and Viability of MC3T3-E1 Cells
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The Annexin V-FITC cell apoptosis detection kit (Beyotime Biotechnology, Shanghai, China) was used to detect cell apoptosis. The MC3T3-E1 cells were incubated with or without DEX and various concentrations of VK2 (10−5, 10−6 and 10−7 M) for 6 days, collected, resuspended in 200 µl of Annexin V-FITC and 10 µl of propidium iodide, and incubated for 20 min at room temperature. Subsequently, flow cytometry was used to evaluate the number of apoptotic cells. The early apoptotic cells are labeled green and the dead and late apoptotic cells are labeled red, while the live cells are not stained.
trypan blue staining was performed to evaluate cell viability. The MC3T3-E1 cells were treated with both DEX and various concentrations of VK2 (10−5, 10−6 and 10−7 M) in FBS-free medium for 6 days and then collected. Ten microliters of trypan blue (Invitrogen, Carlsbad, CA, USA) were mixed with 10 µl of the cell suspension, and 10 µl of the mixture were then added to the cell counting plate. The cell death rates were automatically calculated with a cell counter (Invitrogen). A ReadyProbes® Cell Viability Imaging kit (Life Technologies, Gaithersburg, MD, USA) was also used to detect cell viability at 6 days after the MC3T3-E1 cells were incubated under the different conditions. The blue dye stained all living cells, and the green dye stained the dead cells.
trypan blue staining was performed to evaluate cell viability. The MC3T3-E1 cells were treated with both DEX and various concentrations of VK2 (10−5, 10−6 and 10−7 M) in FBS-free medium for 6 days and then collected. Ten microliters of trypan blue (Invitrogen, Carlsbad, CA, USA) were mixed with 10 µl of the cell suspension, and 10 µl of the mixture were then added to the cell counting plate. The cell death rates were automatically calculated with a cell counter (Invitrogen). A ReadyProbes® Cell Viability Imaging kit (Life Technologies, Gaithersburg, MD, USA) was also used to detect cell viability at 6 days after the MC3T3-E1 cells were incubated under the different conditions. The blue dye stained all living cells, and the green dye stained the dead cells.
9
Cell Viability Assay with Gel Extracts
10
Cell Viability Imaging of Cytokine and α-Syn
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To measure cell viability after cytokine and α-syn treatment, ReadyProbes Cell Viability Imaging Kit (Molecular Probes) was used. Two drops of both NucBlue Live Reagent and NucGreen Dead reagent was added to 1 mL of DMEM/F12, 10% FBS and 1% PSG and a complete media change was performed on the cells. After a 15 min incubation at 37 °C, live imaging of healthy or compromised cells were acquired using the automated fluorescence microscope ImageXpress Micro XLS (Version 5.3.0.1, Molecular Devices, CA, USA) using the 20× (0.45 NA) CFI Super Plan Fluor ELWD ADM objective lens and Lumencor Spectra X configurable light engine source.
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