Transient membrane pore formation during droplet EP was confirmed using the YO-PRO-1 uptake assay, conducted as previously described with some modifications [23] (link), [25] . YO-PRO-1 was purchased from Life Technologies. A 3.0 μl droplet containing 1.0×104 HEK293 cells suspended in DMEM with 10% FBS and 1 μM YO-PRO-1 was added to the silicone oil and droplet EP was performed. The droplet was transferred to 100 μl of DMEM with 1 μM YO-PRO-1 in a 96-well opaque microplate (Immuno Standard Modules Black, Thermo Fisher Scientific). After 1 h incubation at 37 °C, 5% CO2, the fluorescence of the cells was measured by the multimode microplate reader using 485/528 nm (excitation/emission) wavelengths, in accordance with the fluorescence properties of YO-PRO-1. Cell proliferation and YO-PRO-1 uptake after droplet EP were confirmed by transferring the droplet to DMEM/10% FBS/PS and YO-PRO-1 in an 8-well cover glass chamber (Nunc™ Lab-Tek™ II cover glass chamber, Thermo Fisher Scientific) and incubating for 24 h at 37 °C, 5% CO2. The cells were subsequently microscopically observed using an inverted fluorescence microscope (TE-2000U; Nikon) equipped with a 10× objective lens (Plan Fluor; Nikon) and a digital camera (D5000; Nikon).
Yo pro 1
Manufactured by Thermo Fisher Scientific
Sourced in United States, Spain, Germany, Japan
YO-PRO-1 is a fluorescent stain that can be used to detect cell viability. It is a membrane-impermeable dye that can only enter cells with compromised membranes, such as late apoptotic or necrotic cells. YO-PRO-1 binds to nucleic acids and exhibits a green fluorescence upon binding, allowing for the detection of non-viable cells.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (19 mentions)
Propidium iodide, by Thermo Fisher Scientific (15 mentions)
Propidium iodide, by Merck Group (8 mentions)
Sytox green, by Thermo Fisher Scientific (5 mentions)
Facscalibur, by BD (5 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (5 mentions)
Merocyanine 540, by Thermo Fisher Scientific (4 mentions)
Yo pro 1, by Nikon (4 mentions)
Confocal microscope, by Nikon (4 mentions)
Bzatp, by Merck Group (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Yoyo 1, by Thermo Fisher Scientific (4 mentions)
Triton x 100, by Merck Group (4 mentions)
Fluo 4 am, by Thermo Fisher Scientific (4 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (4 mentions)
Ms 222, by Merck Group (3 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
D5000, by Nikon (3 mentions)
Hoechst 33342, by Merck Group (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
171 protocols using yo pro 1
1
Droplet Electroporation and YO-PRO-1 Uptake Assay
2
Apoptosis and Cell Death Assay
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Cells were seeded 15–18 h prior to subsequent treatments. WT hTERT and nuchTERT were submitted to 60 min of 200 µM H2O2 treatment. Cells were allowed to recover in conditioned medium for 24 h. The cells were then harvested and then assayed with YOPRO-1 and PI to monitor apoptosis (YOPRO-1, ThermoFisher Invitrogen, Grand Island, NY, USA ) and cell death (PI) using flow cytometry. In brief, after trysinization, the cells were washed twice with 1 mL of PBS, resuspended in PBS, and then stained with a final concentration of 2.5 µM of YO-PRO-1 (ThermoFisher Invitrogen, Grand Island, NY, USA) and 1 µg of PI (ThermoFisher Invitrogen, Grand Island, NY, USA) for 20 min on ice. Cells were then scored as viable (negative for both markers), apoptotic YOPRO-1 positive/PI-negative or YOPRO-1 positive/PI-positive, and dead (PI positive) while using a BD Biosciences FACS Calibur flow cytometer.
3
Apoptosis Detection with YO-PRO-1
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4
Quantifying RIPK1-Induced Necroptosis
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LDH release was measured in HeLa cells transfected with human RIPK1 (wild type and mutant) to determine necroptosis using the Cytotoxicity Detection Kit (Roche, Switzerland) following the manufacturer’s instructions. The results were expressed as the percentage of total LDH intracellularly present in control cells. Necroptosis was also followed by Yo-Pro-1 uptake, for that HeLa cells transfected with the different RIPK1 plasmids were incubated with 2.5 μM Yo-Pro-1 (Invitrogen, USA) for 5 min, and then fluorescence was determined at 485 ± 9/515 ± 9 nm (excitation/emission) in a Synergy Mx plate reader (BioTek, USA), and the percentage of Yo-Pro-1 was compared to cells treated with 0.1% triton X-100 for 5 min.
5
Spleen Cell YO-PRO-1 Intake Assay
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6
High-Throughput Cytotoxicity Screening and Synergy Analysis
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Cell lines were seeded in 384-well assay plates at a density of 1,000 cells/well in a total volume of 40 μL/well, and incubated at 37°C, 5% CO2 overnight. Following drug exposure, proliferation was measured by staining with Hoechst 33422 (Life Technologies) nuclear dye and apoptosis using YO-PRO-1 (Life Technologies) early apoptosis dye and analyzed using a Thermo CellInsight high content microscope for indicated time. IC50 values were determined using GraphPad Prism 6.0. For drug synergy, fixed dose ratios were used to determine five different drug combinations. Following 72 hours of drug exposure, proliferation and cell death was measured by staining with Hoescht (Life Technologies) nuclear dye and YO-PRO-1 (Life Technologies), respectively, and analyzed using a Thermo CellInsight High Content microscope. Synergistic, additive, or antagonistic effects were determined using the combination index (CI) method of Chou and Talalay41 (link).
7
High-Throughput Cytotoxicity Screening and Synergy Analysis
8
Apoptosis and Necrosis Quantification
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The apoptosis/necrosis assay was performed by double-staining of cells with YO-PRO-1 (Thermo Fisher Scientific, Waltham, MA, USA) and propidium iodide (PI, Sigma-Aldrich) fluorescent dyes. Briefly, HaCaT cells (4.5 × 105) were seeded onto 6-well plates. On the next day, the cells were treated for 24 h with the analyzed compounds at a concentration corresponding to the previously determined MIC50 and MIC99 values. Subsequently, the cells were detached with trypsin (Thermo Fisher Scientific), washed twice with DPBS (1 mL) (Thermo Fisher Scientific), and stained with YO-PRO-1 and PI according to the manufacturer′s protocol for 30 min at 37 °C in dark. The cells were analyzed immediately after staining with 488-nm excitation by FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA), and the data were analyzed by FlowJo software.
9
Apoptosis/Necrosis Assay of Compounds
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Apoptosis/necrosis assay was carried out by double staining of cells with YO-PRO-1 (Thermo Fisher Scientific) and propidium iodide (PI, Sigma-Aldrich) fluorescent dyes. Briefly, HepG2 cells (2 × 105) were seeded onto 6-well plates. The next day, the cells were treated for 24 h with the analysed compounds at a concentration corresponding to half of their IC50 (34 –2.5 µM, 35 –2.3 µM, 36 –2 µM, 37 –2.3 µM)and total IC50 values (34 –5 µM, 35 –4.5 µM, 36 –4 µM, 37 –4.5 µM). Subsequently, the cells were detached with trypsin (Thermo Fisher Scientific), washed twice with DPBS (1 ml) (Thermo Fisher Scientific), and stained with YO-PRO-1 (0.5 µM) and PI (10 µg/ml) according to the manufacturer’s protocol for 30 min at 37 °C in the dark. Immediately after staining, the cells were analysed with 488 nm excitation using a FACSCalibur flow cytometer (Becton Dickinson), and data were analysed using the FlowJo software (Becton Dickinson).
10
Splenic Cell ATP-Induced Apoptosis Assay
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