At the end of each exposure/treatment, H292 cells were trypsinized, counted and seeded in a 96-well (CellCarrier™-96; PerkinElmer #6005550) in triplicate at the density of 1*103 cells/well and then placed in the incubator (5%CO2; 37°C) for 24 h. Then cells were labeled for nuclei (NucBlue™ Live cell Stain, Thermo-Fisher Scientific #R37605), membranes (CellMask™ Green Plasma Thermo-Fisher #C37608) and ACE-2 protein receptor (primary antibody: mouse anti-hACE-2; R&D Systems, #MAB933. Secondary antibody Alexa Fluor™ 546 goat anti-mouse IgG; thermo Fisher Scientific, #A11003). Membrane protein expression was assessed by High Content Screening (HCS) analysis using the PerkinElmer Operetta High-Content Imaging System. A dose-response curve and IC50 for ACE-2 expression were calculated, related to both nicotine in the exposed basal media and puff number. Air exposure and incubator controls were used for all the experiments. HCS analysis of ACE-2 protein expression was evaluated following membrane segmentation. Plates were read under confocal conditions using the 20x long WD objective. Enough fields were imaged to capture at least 500 cells/well. All images were analyzed using Harmony high-content imaging and analysis software (PerkinElmer). Final output values from the analysis were expressed as mean fluorescence intensity (MFI) percentage of control per well.
Cellcarrier 96
Manufactured by PerkinElmer
Sourced in United States
The CellCarrier-96 is a high-quality multiwell plate designed for cell-based assays. It features a 96-well format and is made of polystyrene, providing a reliable and consistent platform for various cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Sp8 confocal microscope, by Leica (3 mentions)
Nucblue live cell stain, by Thermo Fisher Scientific (2 mentions)
Operetta high content imaging system, by PerkinElmer (2 mentions)
Harmony high content imaging and analysis software, by PerkinElmer (2 mentions)
Opera phenix high content screening system, by PerkinElmer (2 mentions)
Alexa fluor 546 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Amqax1000, by Thermo Fisher Scientific (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Mouse α smn, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
F4 80 microbeads, by Miltenyi Biotec (1 mentions)
Hoechst 33342, by Dojindo Laboratories (1 mentions)
Macs ls column, by Miltenyi Biotec (1 mentions)
Doxycycline, by Fujifilm (1 mentions)
Doxycycline, by Nacalai Tesque (1 mentions)
Cellcarrier 384 ultra microplate, by PerkinElmer (1 mentions)
20 protocols using cellcarrier 96
1
Quantifying ACE-2 Expression in H292 Cells
2
Mitochondrial Stress Response Assessment
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Fibroblast cells at 80-90% confluency of a 25 mL flask were seeded on the 96-well plate (CellCarrier-96, PerkinElmer) with a density of 4,000-5,000 cells/well for 24 h. The next day, cells were treated with stress inducers including hydrogen peroxide (H2O2), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), or phenanthroline with the final concentrations as indicated in Table 1 in the culture media for the measurement of mitochondrial ROS, MMP, and fragmentation and length, respectively.Table 1
Preparation of H2O2, FCCP, and phenanthroline concentrations.
| Stress inducers | Reconstituted solution | Stock solution | Final concentrations | Duration of treatment |
|---|---|---|---|---|
| H2O2 (Siribuncha Corp, Lot. No. 0230049) | Water | 3% wt/vol (0.882 M) | 100, 200, and 400 µM | 1 h |
| FCCP (Sigma-Aldrich, | Absolute ethanol | 5,000 µM | 0.01 and 0.1 µM | 1 h |
| Phenanthroline (Sigma-Aldrich, Cat.No.131377) | Absolute ethanol | 25,000 µM | 50 µM | 4 h |
3
Mitochondrial Membrane Potential Quantification
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48 h before the experiment, 1.5 × 104 cells were plated onto optically clear bottom 96-well plates (CellCarrier-96, PerkinElmer). Cells were washed once using PBS and incubated with 1 μM TMRE (Thermofisher) in FDMEM for 45 min at 37°C. Cells were washed once using PBS and nuclei were stained with 1 μg/ml Hoechst in PBS for 10 min, followed by a final wash in PBS. 10 random images were taken using a Leica SP8 confocal microscope as indicated above. TMRE staining per cell was quantified using CellProfiler 4.0.6.
4
Seeding Neurons and Astrocytes in 96-well Plates
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hNs and hpAs were harvested with Accutase (Innovative Cell Technology, Inc, AT104-500), quenched in Neurobasal media, spun 5 min at 1000 rpm at room temperature (RT), passed through a 40 µm filter, and counted using the Countess Automated Cell Counter (Thermo Fisher Scientific, AMQAX1000). Cells were mixed in NBM media to reach a seeding density of 40,000 neurons.cm–2 and 100,000 astrocytes.cm–2 per well. A liquid handling dispenser (Personal Pipettor, ApricotDesigns) was used to uniformly plate the cell mixture in the geltrex-coated 60-inner wells of 96-well plates (PerkinElmer, CellCarrier-96, 6005558).
5
Quantifying Nanoparticle Uptake in Cells
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Cells were seeded in 96-well plates (Cell Carrier™-96; PerkinElmer #6005550) at a density of 2 × 103 cells per well. Nuclei were stained with NucBlue solution (NucBlue™ Live cell Stain, Thermo-Fisher Scientific #R37605) for 15 min at Room Temperature (25 °C) following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). After cell labelling for the nuclei, samples were washed three times in PBS and treated with DMSO, used as vehicle, and different MNPs@MIL[b] and MNP concentrations (5, 10, 15, 20 μg/mL). Cells were imaged using the PerkinElmer Operetta High-Content Imaging System (# HH12000000). Plates were read under confocal conditions using the 63× long WD objective. A specific fluorescence type channel was used to acquire images of NucBlue (Ex: UV light, Em: 460 nm) for nuclei staining, shown in blue. All images were analyzed using Harmony high-content imaging and analysis software (PerkinElmer, Waltham, MA, USA). Initial segmentation of cells was carried out in the DAPI channel by identifying the blue-stained nuclei with an area >30 µm. Finally, the number of spots per Area of Cytoplasm and Nucleus was expressed as mean per well.
6
Quantifying Cell Viability and Death
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Cells were plated at a density of 1 × 104 cells/well in a 96-well plate (CellCarrier-96, PerkinElmer Life Sciences) and allowed to adhere for 4 h. Cells were then transfected with siRNAs as above. After culture for 48 h, live cell numbers (relative to control) were determined by uptake of calcein acetoxymethyl ester (calcein-AM; 2.5 μm , 60 min incubation at 37 °C in HBSS buffer). Fluorescence was measured in a FlexStation 3 (Molecular Devices) with excitation at 490 nm and emission at 520 nm. Numbers of dead cells (as a percentage relative to controls) were determined fluorometrically by propidium iodide uptake (excitation at 535 nm and emission at 617 nm).
7
Quantifying hPSC-Cardiomyocyte Viability
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HiPSC-cardiomyocytes cultured in monolayer in 96 well culture plates for imaging (CellCarrier-96, PerkinElmer) at a density of 30 K cells per well were exposed to 24 hours of anthracyclines at indicated concentrations of the compound. Calcein AM (ThermoFisher) live cell dye in PBS at a final concentration of 4 μM was added to the cells in the well after washing the well 1x with DPBS(-). The dye was incubated with the cells for 30 minutes at 37°C inside a CO2 incubator, followed by microscopic analysis using the EVOS auto II (ThermoFisher) imaging system. Keeping the acquisition settings constant, the Calcein AM fluorescent signal was captured using the GFP filter cube (ThermoFisher, AMEP4951, ex482/25nm; em524/24nm), making 12 images per culture well using a 10x Plan fluorite (NA 0.25) objective (ThermoFisher, AMEP4681). Calcein signal as a fraction of total surface area was then assessed by a custom ImageJ macro, selecting the Calcein signal using a hard (manual) threshold set constant per replicate batch, of which the area was divided over the total area of the image. This fraction represented the area covered by live cells, thus viability of the hPSC-cardiomyocytes.
8
Automated Imaging of Autophagy in Fibroblasts
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48 h before the experiment, 1.5 × 104 cells were plated onto optically clear bottom 96-well plates (CellCarrier-96, PerkinElmer). Cells were treated with 5 mM EGTA or 2 mM CaCl2 for 16 h before the experiment. Cells were washed once using PBS, fixed with 4% (v/v) paraformaldehyde for 12 min at room temperature (RT), permeabilized in phosphate-buffered saline (PBS) containing 0.3% (v/v) Triton X-100 and blocked in 5% (w/v) bovine serum albumin (BSA) for 60 min. Cells were incubated with the following primary antibodies overnight in 4°C: mouse α-SMN (BD Transduction Laboratories), 1:200; rabbit α-LC3B-II (Cell Signaling), 1:200. After washing three times with PBS, cells were incubated with Alexa Fluor secondary antibodies (Invitrogen) for 2 h at RT. Alexa Fluor 647 Phalloidin (ThermoFisher) was added to the secondary antibodies for staining of the fibroblasts’ membrane and segmentation purposes in the open-source software image analysis, CellProfiler 4.0.6 (https://cellprofiler.org/ ) using in-house pipelines. Nuclei were stained with 300 nM 4,6-diamidino-2- phenylindole (DAPI, Molecular Probes). Cells were visualized using a Leica SP8 confocal microscope equipped with a motorised stage for automated acquisition of images, that were acquired at ×20 magnification and ×2.5 digital zoom.
9
Tracking Tumor Macrophages with FF-10832-DiI
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Mice with Capan-1 tumors were administered FF-10832-DiI (4 mg/kg) or the vehicle via the tail vein. The tumors were collected 24 h after administration. Single-cell suspensions of the tumors were prepared using gentle MACS. The single-cell suspensions were purified using F4/80 MicroBeads (Miltenyi Biotec). Briefly, the F4/80-positive cells were magnetically labeled with anti-F4/80 MicroBeads. The cell suspension was then loaded onto a MACS LS Column (Miltenyi Biotec). After removing the column from the magnetic field, the magnetically retained F4/80-positive cells were eluted. The purified cells (2 × 105 cells/well) were seeded into 96-well plates (CellCarrier-96, PerkinElmer, Waltham, MA), and stained for 30 min with 50 nM LysoTracker Deep Red (Thermo Fisher Scientific) in RPMI 1640 containing 10% FBS and 1% Penicillin–Streptomycin. The cells were then stained for 10 min with 10 μg/mL Hoechst 33342 (Dojindo Laboratories, Kumamoto, Japan) in phosphate buffered saline (PBS). The cell images were captured using a confocal quantitative image cytometer CQ1 (Yokogawa Electric, Tokyo, Japan) and analyzed using the CQ1 proprietary measurement software. All experiments were performed in triplicate.
10
Differentiation of Myocytes from iPS Cells
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Screening and mechanistic studies were performed on myocytes differentiated from the iPS cells generated as mentioned above. iPS cells were cultured and maintained as a feeder‐free culture on iMatrix‐511 (892012; Nippi, Tokyo, Japan) in StemFit medium (AK02N; Ajinomoto, Tokyo, Japan) as previously described 14 . Differentiation of MM iPS cells into myocytes was performed by the forced expression of MyoD1 under the control of doxycycline (D5897; Wako, Osaka, Japan) as previously described 12 , 15 . Briefly, on day 0, cells were plated on Matrigel‐coated CellCarrier‐96 or CellCarrier‐384 Ultra microplates (6057300; PerkinElmer, Waltham, MA) with Rock inhibitor (251‐00514; Wako); the next day, medium was switched to Primate ES Cell Medium. On day 2, doxycycline was added to the same medium, and the latter was switched to Minimum Essential Medium Eagle, Alpha Modification (α‐MEM) (21444‐05; Nakalai Tesque, Kyoto, Japan) containing 5% knockout serum replacement (10828028; Thermo Fisher Scientific, Waltham, MA) on day 3. The medium was changed every other day until day 8.
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