Fluorescence images were acquired, analyzed, and quantified using an Axiovert 200 M fluorescence microscope (Zeiss, Oberkochen, Germany) or an automated high-content screening microscope Array Scan VTI (Thermo Fisher, Dreieich, Germany) as described [39 (link),93 (link),94 (link)]. We seeded cells in microscopic dishes (35 mm, MatTek, Ashland, MA, USA) or clear-bottom 96-well plates (Greiner, Kremsmünster Austria) and fixed them with 4% PFA (20 min, RT). For immunofluorescence staining, we additionally permeabilized the cells via incubation with Triton-X 100 (0.1%, 10 min, RT). Antibodies were diluted in 10% FBS/PBS and incubated with samples for 1 h at RT. We washed the cells (n = 3) in PBS and then incubated the samples with fluorophore-labeled antibodies for 1 h at RT. Finally, we stained the nuclei by adding Hoechst 33342 (50 ng/mL in PBS) for 30 min at RT. For automated high-content screening, regions of interest were created using the nucleus signal and each sample was acquired in triplicate, imaging at least 5000 events per sample according to [39 (link)].
Clear bottom 96 well plates
Manufactured by Greiner
Sourced in United States
Clear-bottom 96-well plates are a type of laboratory equipment used for various experimental and analytical purposes. These plates feature a transparent bottom design that allows for visual observation and analysis of samples placed within the individual wells.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m fluorescence microscope, by Zeiss (2 mentions)
Arrayscan vti, by Thermo Fisher Scientific (2 mentions)
Microscopic dishes, by MatTek (2 mentions)
Hk 2 cell, by American Type Culture Collection (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Victor3, by PerkinElmer (1 mentions)
Operetta high content imaging system, by Olympus (1 mentions)
Columbus system, by PerkinElmer (1 mentions)
Scanr imaging system, by Olympus (1 mentions)
Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
Celltiter glo 2.0 cell viability assay, by Promega (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Ko143, by Merck Group (1 mentions)
Duolink kit, by Merck Group (1 mentions)
Sc 8334, by Santa Cruz Biotechnology (1 mentions)
Cellinsight cx5 high content screening platform, by Thermo Fisher Scientific (1 mentions)
Sc 378, by Santa Cruz Biotechnology (1 mentions)
Human recombinant epidermal growth factor, by Thermo Fisher Scientific (1 mentions)
Bovine pituitary extract, by Thermo Fisher Scientific (1 mentions)
14 protocols using clear bottom 96 well plates
1
Automated Microscopy-based Fluorescence Imaging
2
Monitoring Intracellular Calcium Dynamics
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RAW264.7 cells were plated at 40,000 cells/well into black walled, clear bottom 96-well plates (Greiner Bio One, Kremsmünster, Austria) and cultured 2 days up to 90–100% confluence. Before assay, the medium was removed and cells were incubated with FLUO-4 NW for 40 min at 37°C and 5% CO2 as previously described (Molteni et al., 2017 (link)). Fluorescence was monitored every 0.5 s for the preceding 20 s and the 60 s following stimulation using the multilabel spectrophotometer VICTOR3 (Perkin Elmer, Waltham, MA, United States) (excitation, 485 nm; emission, 535 nm). Changes in fluorescence corresponded to changes in intracellular calcium levels. TLQP-21, JMV5656, C3a, and C3a(70-77) were diluted in HBSS and injected into the wells by an automated injector system. Where indicated, antagonists and inhibitors were added at different times before the end of the incubation with FLUO-4 NW as previously described (Molteni et al., 2017 (link)). Briefly: 2 μM TG, 20 min; 2 μM CsA, 15 min; 10 μM U73122, 10 min; 75 μM 2-APB, 15 min; 10 μM SKF-96365, 20 min; 10 μM YM-58483, 20 min; and 1 mM EGTA, 30 min. Before the end of each experiments, cells were stimulated with 10 μM ATP to control for their viability. All experiments were performed at 37°C and fluorescence values (F) were normalized against the baseline acquired immediately before stimulation (F0).
3
High-Content Fluorescence Microscopy Imaging
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Fluorescence images were acquired, analyzed, and quantified using Axiovert 200 M fluorescence microscope (Zeiss) or the automated high-content screening microscope Array Scan VTI (Thermo Fisher) as described [1 (link),33 (link),34 (link)]. We seeded cells in microscopic dishes (35 mm, MatTek) or clear-bottom 96-well plates (Greiner) and fixed them with 4% PFA (20 min, RT). For immunofluorescence staining, we additionally permeabilized the cells via incubation with Triton-X 100 (0.1%, 10 min, RT). Antibodies were diluted in 10% FBS/PBS and incubated with samples for 1 h at RT. We washed the cells (n = 3) in (PBS) and then incubated the samples with fluorophore-labeled antibodies for 1 h at RT. Finally, we stained the nuclei by adding Hoechst 33342 (50 ng/mL in PBS) for 30 min at RT. For automated high-content screening, regions of interest were created using the nucleus signal and each sample was acquired in triplicate, imaging at least 5000 events per sample according to [1 (link)].
4
High-Content Imaging of Cell Cycle
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U-2-OS cells were grown on clear bottom 96-well plates (Greiner) and either pre-extracted with cold 0.5% Triton X-100 CSK buffer for 5 min before fixation or directly fixed in 4% formaldehyde for 10 min. For EdU (5-ethynyl-2′-deoxyuridine) labeling, cells were incubated with 40 μM EdU for 20–30 min. EdU was detected using Click-iT Plus EdU Kit for Imaging (C10640). Plates were imaged on a Perkin Elmer Operetta high-content imaging system or Olympus Scan-R imaging system using a 20x objective. 35 fields per well were imaged, and ∼2,000 cells per condition were analyzed. Single-cell fluorophore intensities were extracted using the Columbus system (Perkin Elmer) or Scan-R analysis software. Cell cycle phases were gated based on DAPI and EdU or PCNA intensities. Graphs were generated using Tableau 2019.3 and GraphPad Prism.9 software.
5
Optogenetic T Cell Activation Assay
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A 20 µg/mL solution of PhyB protein was added to washed streptavidin-coated polystyrene beads and then incubated for 1 h in ice. After washing with PBS supplemented with 0.5 mM TCEP, the beads were resuspended in complete DMEM/F-12 medium. The beads were distributed in black clear-bottom 96-well plates (Greiner). Plates were illuminated on optoPlate for 3 min under 780 nm light to ensure a closed conformation of all PhyB proteins. Primary T cells labeled with 0.3 µg/mL of LiTE protein were added at a concentration of 5 × 105 cells/well in the presence of 2 µg/mL anti-CD28 antibody. The plates were incubated on the optoPlate located in the cell culture incubator and exposed to the indicated photostimulation programs. The harvested T cells were then analyzed by flow cytometry to measure cell surface protein expression and by ELISA of their supernatants to measure IL-2 secretion. Plates were read at 450 nm with a Tecan INFINITE M1000 PRO. For T cell proliferation analysis, T cell were labeled with the CellTrace Violet Cell Proliferation Kit (Thermo Fischer Scientific) for 20 min at 37 °C, before being stimulated. After 72 h of illumination at 630 nm or 780 nm, the CellTrace Violet dilution was analyzed by flow cytometry.
6
Cell Viability Assay in U-2-OS
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U-2-OS were transfected with siRNAs and after 24 h seeded in clear bottom 96-well plates (Greiner) at 500 cells/well in triplicates. Cell viability was measured every 24 h for 4 d using CellTiter-Glo 2.0 Cell Viability Assay (G9242; Promega) according to manufacturer’s recommendations.
7
Cellular Fluorescence Quantification
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Black-sided, clear-bottom 96-well plates (Greiner) were incubated for 1 h in 10μg/ml poly-L -lysine (Sigma) before cells were seeded at 40000 cells per well. Plates were incubated overnight at 37°C and 5% CO2 before media was replaced with phenol red-free DMEM containing transport substrates [8 μM MX, rhodamine 123 (R123) or pheophorbide A (PhA)] in the presence or absence of 0.5 μM Ko143 (Sigma). Cells were incubated for 1 h at 37°C and were subsequently washed once in PBS. Cells were incubated for a further 1 h at 37°C in phenol red-free DMEM alone, supplemented with Ko143 where required. Cells were washed with ice-cold PBS, before incubation with paraformaldehyde [PFA, 4% (w/v), 15 min] and two final washes with PBS. Cellular fluorescence was determined using a fluorescence plate reader (MDC Flexstation). Fluorescence data were corrected for values obtained from incubations with 1% v/v DMSO, which was the maximum solvent concentration used.
8
In situ PLA for Protein Interactions
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In situ PLA was performed using a Duolink kit (Sigma-Aldrich, cat. DUO92101) following the manufacturer’s protocol. HEK293 cells were cultured on clear bottom 96 well plates (Greiner Bio-One), transfected with the required plasmids and fixed with 4% PFA. After washing the plate 3 times with PBS, the cells were blocked (1% BSA + 0.075% Triton X100) for 1 h at room temperature and then incubated with primary antibodies overnight. Antibodies included: mouse anti-Hsp20 (SC-51955, SantaCruz, 1:500), mouse anti-GFP (632381, Living colors, 1:500), rabbit anti-GFP (SC-8334, SantaCruz, 1:1000), and mouse anti-Gβγ (SC-378, SantaCruz, 1:1000). After removal of primary antibodies, the samples were incubated with anti-mouse PLUS and anti-rabbit MINUS PLA probes for 1 h at 37 °C. Subsequent steps of ligation and amplification were according to the manufacturer’s protocol. After the last wash, cells were stained with DAPI (1 μg/ml) and imaged using an epifluorescence high content imaging microscope with a 20X objective (CellInsight CX5 High Content Screening platform, ThermoFisher). Data analysis was performed using FACS analysis software (FlowJo, USA). To prevent false positives, cells with saturating YFP fluorescence or less than 4 positive signals (dots) per cell were not considered in the analysis.
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In situ PLA was performed using a Duolink kit (Sigma-Aldrich, cat. DUO92101) following the manufacturer’s protocol. HEK293 cells were cultured on clear bottom 96 well plates (Greiner Bio-One), transfected with the required plasmids and fixed with 4% PFA. After washing the plate 3 times with PBS, the cells were blocked (1% BSA + 0.075% Triton X100) for 1 h at room temperature and then incubated with primary antibodies overnight. Antibodies included: mouse anti-Hsp20 (SC-51955, SantaCruz, 1:500), mouse anti-GFP (632381, Living colors, 1:500), rabbit anti-GFP (SC-8334, SantaCruz, 1:1000), and mouse anti-Gβγ (SC-378, SantaCruz, 1:1000). After removal of primary antibodies, the samples were incubated with anti-mouse PLUS and anti-rabbit MINUS PLA probes for 1 h at 37 °C. Subsequent steps of ligation and amplification were according to the manufacturer’s protocol. After the last wash, cells were stained with DAPI (1 μg/ml) and imaged using an epifluorescence high content imaging microscope with a 20X objective (CellInsight CX5 High Content Screening platform, ThermoFisher). Data analysis was performed using FACS analysis software (FlowJo, USA). To prevent false positives, cells with saturating YFP fluorescence or less than 4 positive signals (dots) per cell were not considered in the analysis.
9
Toxicity Evaluation of Chemicals in HK-2 Cells
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HK-2 cells were purchased from ATCC (Manassas, VA, USA) and maintained in Keratinocyte-SFM media containing 10% Fetal Bovine Serum, 50 mg/L Bovine Pituitary Extract and 5 µg/L human recombinant Epidermal Growth Factor (all from Gibco, Waltham, MA, USA) as described previously (Mossoba et al., 2016a (Mossoba et al., , 2016b)) . In preparation for treatment exposures for the duration of 1, 2, 4, 8, and 16 days, cells were seeded in clear-bottom black-or white-wall (for fluorescence or luminescence assays, respectively), clear-bottom 96-well plates (Greiner, Frickenhausen, Germany) at a density of 2 x 10 5 , 1 x 10 5 , 0.5 x 10 5 , 0.25 x 10 5 , and 0.125 x 10 5 cells per mL, respectively. Serially diluted concentrations of 3-monochloropropane-1,2-diol (3-MCPD; 0-100 mM), phenylmercuric acetate (PMA; 0-100 µM) (positive control), or valproic acid (VAL; 0-100 mM) (negative control) were prepared in HK-2 media. All treatment compounds were purchased from Sigma (St. Louis, MO, USA) and added to appropriate wells for the intended exposure duration and kept at 37°C in a humidified incubator with 5% CO 2 before performing toxicity assays.
10
Cytotoxic Effect of Methylamphetamine on HK2 Cells
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HK2 cells (human kidney proximal tubular cells) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HK2 cells were cultured in commercial keratinocyte medium containing 10% fetal bovine serum (FBS; HyClone), 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere with 5% CO2 at 37 °C. In preparation for treatment of MA (Sigma, St. Louis, MO, USA), cells were seeded in clear-bottom 96-well plates (Greiner, Frienckenhausen, Germany) at a density of 1 × 105. Serially diluted concentrations of MA were prepared in MA in HK-2 media. All treatments were added to appropriate wells for the intended exposure duration and kept at 37 °C in a humidified incubator with 5% CO2 before performing experiments. For the in vitro study, HK2 cells were induced with MA at 2 mM and 5 mM for 24 h separately. To evaluate the cytotoxic effect of MA, the MTT assay was used to measure cell viability in MA-treated and vehicle-treated HK-2 cells.
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