HepG2 cells were seeded at a density of 3×104 cells/well in a black 96-well clear bottom plate (Greiner Bio One, Monroe, NC). The next morning, media was aspirated, and cells were washed twice with Hank’s Balanced Salt solution HBSS (Thermo Fischer Scientific, Waltham, MA). 25 μM 100 μL 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) dye in HBSS was added to each well. After a 45-minute incubation, DCFDA was removed, cells were washed 1x with HBSS and the appropriate treatment diluted in complete medium without phenol red was added. Fluorescence intensity at Ex/Em 485/535 nm was measured using a Synergy H1 plate reader (BioTek, Winooski, VT).
96 well black clear bottom plate
Manufactured by Greiner
Sourced in Austria
96-well black/clear bottom plates are a type of laboratory equipment used for various applications. These plates have 96 individual wells, with black side walls and a clear bottom. They are designed to facilitate optical-based measurements and assays, such as fluorescence and absorbance detection, in a multi-well format.
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Lab products found in correlation
Poly l ornithine laminin, by Merck Group (2 mentions)
Ecfp tevs ypet, by Addgene (2 mentions)
Spectramax i5, by Molecular Devices (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Synergy neo2 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions)
Fluo 8, by AAT Bioquest (1 mentions)
A23187, by Merck Group (1 mentions)
M1000 pro, by Tecan (1 mentions)
Fish gelatin, by Merck Group (1 mentions)
4 6 diamidin 2 phenylindol dapi, by Merck Group (1 mentions)
Mouse anti rabbit igg fitc, by Santa Cruz Biotechnology (1 mentions)
Mouse anti pax6, by Abcam (1 mentions)
Mouse anti trk b, by Santa Cruz Biotechnology (1 mentions)
Mouse anti nestin, by Santa Cruz Biotechnology (1 mentions)
Axiovert 200m fluorescence microscope, by Zeiss (1 mentions)
Celltiter glo kit, by Promega (1 mentions)
Vimentin, by Agilent Technologies (1 mentions)
On targetplus smartpool sirna, by Horizon Discovery (1 mentions)
17 protocols using 96 well black clear bottom plate
1
Oxidative Stress Quantification in HepG2 Cells
2
Ara-TM and DN-AraTM Dimerization Assays
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Ara-TM and DN-AraTM dimerization assays were conducted as previously described [38 (link),39 (link)]. The constructs in the pAraTMwt plasmids and the reporter plasmid (pAraGFPCDF) were co-transformed with or without the pAraTMDN constructs for the Ara-TM homodimerization and DN-AraTM heterodimerization assays respectively into the AraC-deficient E. coli strain SB1676 and streaked onto selective LB plates (100 μg/mL ampicillin, 50 μg/mL kanamycin, and 100 μg/mL spectinomycin). Colonies were picked for each construct and grown in 2 mL of selective lysogeny broth (LB) for 12 h at 37 °C and 250 rpm. Cultures were then diluted into selective media with 1 mM isopropyl β-d -1-thiogalactopyranoside (IPTG) for protein induction and grown in a 2.0-mL-deep, 96-well PP plate (PlateOne) for additional 6 h at 37 °C and 250 rpm. We then transferred 200 μL of each culture to a black 96-well, clear bottom plate (Greiner). Absorbance measurements at 600 nm as well as GFP fluorescence emission measurements at 530 nm after excitation at 485 nm were collected using a Synergy Neo2 Hybrid Multi-Mode Microplate Reader (Biotek). The results are reported as the ratio of fluorescence emission at 530 nm to absorbance at 600 nm.
3
Calcium Mobilization Assay in NPCs
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Kinetic measurements of transient intracellular calcium mobilization in NPCs were performed in poly-L-ornithine/laminin (Sigma–Aldrich) coated black 96-well clear-bottom plates (Greiner Bio-One). Cells were seeded at a density of 3.0 × 105 cells per ml and incubated for 24 h. The following day, the medium was removed and cells were loaded with 4 µM Fluo-8 (AAT Bioquest) as the indicator dye, reconstituted in a modified Tyrode’s assay buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 135 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, and 10 mM glucose adjusted to pH = 7.4.) and incubated for 30 min at 37 °C and 5% CO2, and another 30 min at room temperature in the dark [27 (link),28 (link)]. Then, the Fluo-8 loading buffer was removed and replaced with fresh Tyrode’s assay buffer. The agonists, diluted in Tyrode’s assay buffer, were added automatically using the injector unit from Tecan Infinite M1000 Pro microplate reader Tecan (Maennedorf, Switzerland). After 16 s of baseline measurements, the compound was added, and fluorescence was measured for 80 s using excitation at λ = 490 nm and emission at λ = 525 nm. Responses were measured as the maximal peak height in relative fluorescent units (RFUs), and the maximum fluorescence signal was generated with the calcium ionophore A23187 (Sigma–Aldrich).
4
Immunocytochemistry of Neural Progenitor Cells
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NPCs were seeded at a density of 3 × 104 cells per ml in poly-L-ornithine/laminin (Sigma–Aldrich) coated black 96-well clear-bottom plates (Greiner Bio-One) and allowed to attach overnight. Cells were fixed and permeabilized with pre-chilled methanol at −20 °C for 10 min. After blocking with 0.5% fish gelatin (Sigma–Aldrich) in PBS, cells were incubated with the primary antibody overnight at 4 °C, followed by secondary antibody (1:100) for 2 h at room temperature. Primary antibodies used were as follows: mouse-anti-Nestin (1:100, Santa Cruz biotechnology, Dallas, TX, USA, sc-23927), mouse-anti-TrkB (1:100, Santa Cruz Biotechnology, sc-377218), mouse-anti-Pax6 (1:400, Abcam, Cambridge, MA, USA, ab5790), and rabbit-anti-GluA1 (1:100, Merck Millipore, Darmstadt, Germany, A1504). Secondary antibodies were purchased from Santa Cruz Biotechnology (mouse anti-rabbit IgG-FITC (fluorescein isothiocyanate), sc-2359; goat anti-rabbit IgG-Rhodamine, sc-2091; mouse IgG kappa binding protein (m-IgGκ BP-CFL) 488, sc-516176). For nuclear staining, cells were incubated with 4′,6-Diamidin-2-phenylindol (DAPI, Sigma–Aldrich) for 15 min at room temperature at a concentration of 3 µM. Images were taken with the Axiovert 200M fluorescence microscope (Zeiss, Jena, Germany).
5
Quorum Sensing Signaling Assay
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6.4 μM stocks of 3OC6-HSL or C8-HSL were serially diluted in SWTO medium to the final concentrations indicated. An overnight culture of the strain being tested was diluted 1:100 into 200 μl of SWTO in each well of a 96-well black clear-bottom plate (Greiner Bio-One, Monroe, NC). Treatments were arranged, and blank wells were included, to minimize potential light contamination between treatments skewing results. Specifically, final concentrations of 0, 12.5, 25, 100, 400, 800, 1600, and 3200 nM of C8-HSL were used in consecutive horizontal rows, and the same concentrations of 3OC6-HSL were placed in consecutive vertical columns, with the placement of C8-HSL and 3OC6-HSL reversed for the assays with DC20 and DC22. Blank columns were included after the first four columns to help minimize light contamination. Plates were incubated at 24°C and while shaking at 200 rpm, and the optical density at 600 nm (OD600) and luminescence were measured in a Synergy 2 plate reader (BioTek, Winooski, VT). OD600 readings were divided by 0.46 to correspond to the optical density of a 1-cm path-length, and relative luminescence was normalized to the path-length corrected OD600 of ~1.0 to give specific luminescence for each experimental condition.
6
CD4 DARPin-gp140 Binding Assay
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Assays are performed as described previously (Chand et al., 2017) . Briefly, purified CD4
DARPin variants and gp140 proteins were coated onto 96-well black, clear-bottom plate (Greiner), 15 pmoles per well, for 1 hour. The wells were subsequently washed 3 times with blocking buffer (1mM MnCl2, 0.1mM CaCl2, 10mM HEPES, 150mM NaCl, and 10% FBS) and then incubated with the blocking buffer for 1 hour. Wells were then washed 3 times with wash buffer (1mM MnCl2, 0.1mM CaCl2, 10mM HEPES, 150mM NaCl, and 1% FBS). A3.01 (CD4+), A2.01 (CD4-
), Tzmbl and J-Lat 10.6 full length cells (50 µl/well of 4×10 6 cells per ml) were added in cell dilution buffer (wash buffer containing 5% FBS) and allowed to bind for 1 hour. Wells were then washed 5 times with wash buffer and the remaining bound cells were detected with the CellTiterGlo kit (Promega) as per the manufacturer's instructions.
DARPin variants and gp140 proteins were coated onto 96-well black, clear-bottom plate (Greiner), 15 pmoles per well, for 1 hour. The wells were subsequently washed 3 times with blocking buffer (1mM MnCl2, 0.1mM CaCl2, 10mM HEPES, 150mM NaCl, and 10% FBS) and then incubated with the blocking buffer for 1 hour. Wells were then washed 3 times with wash buffer (1mM MnCl2, 0.1mM CaCl2, 10mM HEPES, 150mM NaCl, and 1% FBS). A3.01 (CD4+), A2.01 (CD4-
), Tzmbl and J-Lat 10.6 full length cells (50 µl/well of 4×10 6 cells per ml) were added in cell dilution buffer (wash buffer containing 5% FBS) and allowed to bind for 1 hour. Wells were then washed 5 times with wash buffer and the remaining bound cells were detected with the CellTiterGlo kit (Promega) as per the manufacturer's instructions.
7
Profiling SARS-CoV-2 NSP5 Protease Cleavage
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Predicted NSP5 cleavage sites were cloned into ECFP-TevS-YPET (Addgene Plasmid #100097) [103 (link)] Briefly, the plasmid was re-cloned to put the Tev Protease Site between an XbaI site and a BsiWI site. Cleavage sequences were cloned in between XbaI and BsiWI sites, restoring the XbaI and BsiWI sites, with one Glycine on each side of the predicted NSP5 cleavage sequences. This approach was based on a cloning strategy used previously to study norovirus protease cleavage sites [104 (link)]. For protease cleavage assay, 3x10^4 HEK 293T cells were plated in DMEM + 10% FBS into 96 well black, clear bottom plates (Greiner). 24 hours later, cells were transfected in quadruplicate with 0.1ug of FRET plasmid containing the NSP5 cut-site and 0.1ug of either WT-NSP5 or mutant NSP5C145A expression plasmids [17 (link)] with Lipofectamine 3000 following manufacturer protocol for 96-well plates. 24 hours later, media was removed and PBS was added to wells, and wells were imaged on a Spectramax i5 instrument (Molecular Devices) with the following wavelengths: 420/485 nm for ECFP, 485/535 nm for YPET and 420/535 nm for FRET as previously described [103 (link)]. After background subtraction of un-transfected wells, FRET efficiency was calculated as FRET/ECFP.
8
Quantifying SARS-CoV-2 NSP5 Protease Cleavage
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Predicted NSP5 cleavage sites were cloned into ECFP-TevS-YPET (Addgene Plasmid #100097)(89 (link)) Briefly, the plasmid was re-cloned to put the Tev Protease Site between an XbaI site and a BsiWI site. Cleavage sequences were cloned in between XbaI and BsiWI sites, restoring the XbaI and BsiWI sites, with one Glycine on each side of the predicted NSP5 cleavage sequences. This approach was based on a cloning strategy used previously to study norovirus protease cleavage sites(90 (link)). For protease cleavage assay, 3e4 HEK 293T cells were plated in DMEM + 10% FBS into 96 well black, clear bottom plates (Greiner). 24 hours later, cells were transfected in quadruplicate with 0.1ug of FRET plasmid containing the NSP5 cut-site and 0.1ug of either WT-NSP5 or mutant NSP5C145A expression plasmids (16 (link)) with Lipofectamine 3000 following manufacturer protocol for 96-well plates. 24 hours later, media was removed and PBS was added to wells, and wells were imaged on a Spectramax i5 instrument (Molecular Devices) with the following wavelengths: 420/485 nm for ECFP, 485/535 nm for YPET and 420/535 nm for FRET as previously described(89 (link)). After background subtraction of un-transfected wells, FRET efficiency was calculated as FRET/ECFP.
9
Immunofluorescence Analysis of Tumor Cell Markers
10
Quantifying ROS in ARPE-19 Cells
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ARPE-19 cells were seeded into 96-well black/clear bottom plates (Greiner Bio-One, Kremsmünster, Austria) for the determination of Reactive Oxygen Species (ROS). The 2′,7′-dichlorofluorescein diacetate (DCFDA)-Cellular ROS Detection Assay kit was used for the detection (Abcam, Cambridge, UK). DCFDA, which is initially non-fluorescent, is oxidized to DCF, a highly fluorescent molecule, and its intensity was measured using the BioTek Cytation cell imaging multimode microplate reader (BioTek U.S., Winooski, VT, USA) in end point mode at Ex/Em = 485/535 nm. The microplate reader’s imaging acquisition mode enabled the capture of fluorescence pictures of each well, which were utilized to calculate the number of cells.
ROS generation was then represented as a percentage relative to the control as the fluorescence intensity normalized to the number of cells.
ROS generation was then represented as a percentage relative to the control as the fluorescence intensity normalized to the number of cells.
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