Ulk1/2+/+ and Ulk1/2−/− MEFs stably expressing pGF1-GAS-LUC were plated in a 96-well plate (three or five replicates of 5000 cells per well) and 24 hours later were maintained in serum-free RPMI 1640 medium overnight. Serum-starved cells were left untreated (control) or were treated with 2.5×103 IU/ml of mouse IFNγ for 6 hours and then lysed using 1x Reporter Lysis Buffer (Promega). Luciferase assay substrate buffer (Luciferase Assay System # E4030, Promega) was used per the manufacturer’s instructions and luciferase activities were measured using a Cytation 3 cell imaging multi-mode microplate reader (BioTek).
Cytation 3 cell imaging multi mode microplate reader
Manufactured by Agilent Technologies
Sourced in United States
The Cytation 3 cell imaging multi-mode microplate reader is a laboratory instrument designed for automated cell imaging and measurement. It is capable of capturing high-quality images and performing quantitative analysis of various cellular parameters within microplates.
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7 protocols using cytation 3 cell imaging multi mode microplate reader
1
Luciferase Assay for IFNγ Response
2
Immunofluorescence Assay for AMPK and pAMPK
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For AMPK and phospho-AMPK (pAMPK) detection, an immunofluorescence assay was carried out. Cells were washed with PBS, fixed for 15 min in 4% paraformaldehyde, permeabilized in 0.25% Triton X-100 for 5 min, and washed with PBS. After blocking in 4% goat serum at room temperature for 1 h, the cells were washed and incubated overnight at 4°C with mouse monoclonal to AMPK alpha 1 + AMPK alpha 2 antibodies (Abcam, Cambridge, UK) or rabbit monoclonal phospho-AMPKα (Thr172) antibodies (Cell Signaling Technology, Danvers, MA, USA). Cells were washed 3 times with PBS and incubated at ambient temperatures for 6 hours with Hoechst staining (2.5 μg/mL) together with each corresponding polyclonal secondary antibody, goat anti-rabbit IgG CF 594, or goat anti-mouse polyvalent immunoglobulins (G,A,M)-FITC, all from Sigma-Aldrich (St. Louis, MO, USA). Cells were washed 3 times with PBS and read with a Cytation 3 cell imaging multi-mode microplate reader (Biotek Instruments, Winooski, VT, USA). AMPK was detected by measuring the fluorescence with excitation at 490 nm and emission at 520 nm for AMPK and with excitation at 590 nm and emission at 620 nm for pAMPK. To ensure that the extracts/fractions were not cytotoxic, the cell count was performed by taking microphotographs of the Hoechst-stained cells at 4x, using the DAPI imaging filter cube.
3
Fluorescence Analysis of Cooked Crayfish
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The intrinsic fluorescence intensities of the cooked crayfish samples were determined by fluorescence spectroscopy using a Cytation 3 Cell imaging multimode microplate reader (BioTek Instruments, Winooski, USA). The concentration of MPs was adjusted to 500 μg/mL and placed in an opaque 96-well plate. The excitation wavelength was 280 nm and the emission spectra measured in the range of 300–430 nm. The excitation and emission slit widths were 2.5 nm, and the scanning speed was 2 nm/s.
4
Hypertrophic Adipocyte Model for Obesity Research
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The 3T3-L1 preadipocyte cell line was cultured and differentiated as described elsewhere [23 (link)]. Hypertrophied adipocytes were obtained using high glucose medium containing insulin (4.5 mg/mL) after differentiation [24 (link)]. Under these conditions, cells become hypertrophic adipocytes, a cell model characterized by a high level of cytoplasmic lipid accumulation, insulin-resistance, and exacerbated oxidative stress, a situation reasonably similar to that of obese adipose tissue [16 (link),24 (link)]. On day 18, cells were treated with the respective extract or fractions, which were dissolved in 10% FBS/high glucose (4.5 mg/mL) DMEM medium with 1 μM insulin and maintained for 48 h. For cell treatments, extract or fractions were reconstituted in cell culture medium plus DMSO at a maximum final concentration of 0.3% v/v after solvent evaporation. The crystal violet assay was performed as reported to dismiss the possible cytotoxic effects of the extract/fractions at the working concentrations [24 (link),25 ]. Quantification of intracellular lipid droplets in hypertrophic adipocytes was performed as reported [24 (link)] using the staining AdipoRed Assay Reagent and the Cytation 3 cell imaging multi-mode microplate reader (Biotek Instruments, Winooski, VT, USA).
5
Evaluation of Caspase-3/7 Activity Assay
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To assess the activity and catalytic function of caspase3/7 (DEVD-ase), a Cayman fluorescence assay kit was used based on our adopted established protocol [10 (link),31 (link),48 (link)]. Briefly, the cells were seeded in 96-well plates to reach about 50% confluence, to prevent high non-specific fluorescence, and were then treated with TMZ (100 µM), Simva (1 and 2.5 µM for U251 and U87, respectively), ASH (1.5 µM), TMZ/Simva and TMZ/Simva/ASH, for 48 h based on our experimental protocol (OEP). For the combinations, Simva was pretreated for 4 h. All reagents were freshly prepared, including the assay buffer, caspase3/7 substrate, active caspase-3 positive control and caspase3/7 inhibitor solution. The plates were centrifuged at 800× g for 5 min, followed by aspiration of the supernatant, addition of the assay buffer and another centrifugation. Later, lysis buffer was added, followed by orbital shaking and centrifuging (800× g). Caspase3/7 inhibitor, active caspase-3 positive control and caspase3/7 substrate solutions were then added (30 min, room temperature, and a dark place). The fluorescence intensity of each well was measured by BioTek Cytation 3 cell imaging multi-mode microplate reader (Biotek Cytation 3, USA) (excitation wavelength: 485 nm, emission wavelength: 535 nm).
6
Evaluating PHB-PEI Nanoparticle Cytocompatibility
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To assess PHB-PEI NPs cytocompatibility, a Cell Counting Kit-8 (CCK-8) assay was carried out. Briefly, PC3, Caco-2, MCF-7, and MCF10A cells were seeded in a 96-well plate in 100 μL at a density of 4 × 103 cells/well 24 h before treatment. After 24 h, the cells were treated with 10 μL of PHB-PEI NPs at different concentrations (25–250 μg/mL) and incubated at 37 °C for 24–72 h. Then, 10 μL of CCK-8 solution were added to each well, and the plate was then incubated under cell culture conditions for 1–4 h. The optical density of formazan salt at 450 nm was measured using a Cytation 3 Cell Imaging Multi-Mode microplate reader (Biotek, Milan, Italy)). PHB-PEI NPs cytocompatibility was expressed as a percentage relative to the control and calculated by the equation:
where OD sample is the optical density of cells treated with PHB-PEI NPs and OD control is the optical density of untreated cells.
where OD sample is the optical density of cells treated with PHB-PEI NPs and OD control is the optical density of untreated cells.
7
Genome-wide HCMV Replication Screening
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The Dharmacon SMARTpool human druggable genome (G-004600-05), cell cycle (G-003250-02), and protein kinase (G-003500-02) siRNA libraries against 6,881 gene targets were prepared in the 96-well format at a concentration of 3 μM (diluted in Thermo Scientific siRNA buffer) at the Division of Infection and Pathway Medicine, University of Edinburgh, followed by the set-up of 384-well master plates at a concentration of 311 nM, using a RapidPlate 384 liquid handling robot (Qiagen). The complete protocol can be found in reference 38 (link). Low-passage NHDF cell suspension (at 3,000 cells per well) were reverse transfected with siRNA and Lipofectamine RNAiMAX (Invitrogen) using MultiDrop 384. At 48 h posttransfection, media were removed, and cells were infected with strain TB40/E-GFP at a multiplicity of infection (MOI) of 5. Absolute GFP expression is measured by Cytation 3 cell imaging multimode microplate reader (BioTek) every 24 h for 7 days, and relative GFP expression (or replication ratio) was calculated by dividing the absolute GFP expression of infected cells with the corresponding gene knockdown by the nontargeting siRNA control sample.
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