The harvested cells were lysed in ice-cold reporter lysis buffer. The lysate was centrifuged at 12,000×g for 10 min. Fluorescence intensity of GFP in 100 µl supernatant was measured using a VICTOR X5 2030 Multilabel Plate Reader (PerkinElmer) at an excitation wavelength of 488 nm and emission wavelength of 535 nm. The autofluorescence of the uninfected lysate was detected as background and subtracted from measurements of the EGFP-STIV-infected lysates.
Victor x5 2030 multilabel plate reader
Manufactured by PerkinElmer
Sourced in United States
The Victor X5 2030 Multilabel Plate Reader is a high-performance microplate reader designed for a wide range of applications in life science research. It is capable of performing absorbance, fluorescence, luminescence, and time-resolved fluorescence measurements on microplates, allowing researchers to conduct various assays and analyses.
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Lab products found in correlation
96 well plate, by Greiner (2 mentions)
Cell titer aqueous one solution, by Promega (1 mentions)
Caspase glo assay kit, by Promega (1 mentions)
Celltiter glo, by Promega (1 mentions)
Vi cell cell viability analyzer, by Beckman Coulter (1 mentions)
Cisplatin, by Cayman Chemical (1 mentions)
Caspase glo 3 7, by Promega (1 mentions)
6 protocols using victor x5 2030 multilabel plate reader
1
Measuring EGFP-STIV Fluorescence in Lysates
2
KLHL14 Depletion Impacts Cell Proliferation
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To assess proliferation, we performed MTS colorimetric assays in KLHL14-depleted and control-transfected NCI-H2052 and NCI-H2452 cells, stimulated or not with TGF-β. Briefly, 2 × 103 cells/well were plated into 96-well microplates and starved for 24 h prior to TGF-β exposure. CellTiter® AQueous One Solution (Promega Corporation Madison, WI, USA) was used following supplier's guidelines. Cell growth was determined by the amount of formazan produced, analyzed by measuring the absorbance at 492 nm in a PerkinElmer VICTOR X5 2030 Multilabel plate reader (PerkinElmer, Waltham, MA, USA). The analysis was carried out at least in triplicates and the experiments performed 3 times.
For colony formation assay, 1 × 103 NTC or KLHL14-depleted cells/well were seeded onto 6-well plates in complete media, allowed to attach and incubated until colonies were formed. Approximately 6–8 days after, colonies were subsequently washed with PBS, fixed with absolute methanol, and stained at room temperature for 30 min with crystal violet solution prepared at 0.5% in 25% methanol and 75% distilled water. Colonies were then additionally washed with PBS, dry and counted manually for statistical analysis.
For colony formation assay, 1 × 103 NTC or KLHL14-depleted cells/well were seeded onto 6-well plates in complete media, allowed to attach and incubated until colonies were formed. Approximately 6–8 days after, colonies were subsequently washed with PBS, fixed with absolute methanol, and stained at room temperature for 30 min with crystal violet solution prepared at 0.5% in 25% methanol and 75% distilled water. Colonies were then additionally washed with PBS, dry and counted manually for statistical analysis.
3
Evaluating Cell Viability with Resazurin
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We determined cell viability by the resazurin reagent. For this purpose, CO 88BV59-1 and normal B cells (1×105) were added to each well in 96-well culture plates treated with vincristine (0.05–50 μM) and crocin (0.2–200 μM) up to 72 h. Next, 20 μl of the resazurin reagent was added to each well, and the plates were incubated for 4 h. The fluorescence intensity of the product resorufin, proportional to the number of viable cells per well, was measured via a fluorescence Victor X5 2030 Multilabel Plate Reader (Perkin Elmer, Shelton, Connecticut) with excitation at 530 nm and emission at 590 nm.16 (link)
4
Caspase Activity Measurement in Liver
5
Cell Viability Assay for Drug Screening
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4,000 cells/well (2,000 cells/well for A204/DasR) were seeded into a 96-well plate (Greiner Bio-One, Kremsmunster, Austria) and incubated for 24 hr at 37°C, 5% CO2. After that, medium (5% FBS) containing drugs or vehicle controls was added to the cells and incubated for an additional 72 hr. Cell viability was measured using CellTiter-Glo (Promega), using a Victor X5 2030 Multilabel plate reader (Perkin Elmer). Cisplatin was obtained from Cayman Chemical Company, doxorubicin from LC Labs, and rapamycin from Calbiochem (San Diego, CA, USA). Additional growth assays were carried out using a ViCell Cell Viability Analyzer (Beckman Coulter). Briefly, 125,000 cells were seeded on 60-mm dishes in media containing 10% FCS. Cells were treated for 3–5 days with different drug concentrations in media containing no serum. Following treatment, viability was assessed using the trypan blue exclusion method. Each condition has been measured in triplicate.
6
Caspase 3/7 Activity Assay
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4,000 cells/well were seeded into a 96-well plate (Greiner Bio-One, Kremsmunster, Austria) and incubated for 24 hr at 37°C, 5% CO2. After that, medium (5% FBS) containing drugs or vehicle controls was added to the cells and incubated for an additional 48 hr. Caspase 3/7 activity was measured using Caspase 3/7 Glo (Promega), with a Victor X5 2030 Multilabel plate reader (Perkin Elmer).
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