3T3-L1 adipocytes (Day 12) grown in cell culture plates were rinsed twice with PBS. Cell lysis buffer (1 M Tris-HCl pH 7.5, 1 M MgCl2, and 10% Triton X100) was added to each well, and the cells were harvested into an Eppendorf tube using a cell scraper. The cells were disrupted with an ultrasonicator, and the triglyceride and protein levels in the cell lysates were measured. The triglyceride concentrations were determined using the Triglyceride E test kit (Wako), and the absorbance was measured at 595 nm using a PowerScan4 (Biotek) plate reader. Protein concentration was determined using Bio-Rad DC Protein Assay (Bio-Rad) reagents, and the absorbance was measured at 595 nm using a PowerScan4 (Biotek) plate reader. The triglyceride content of each sample was normalized to its corresponding protein content.
Powerscan4
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The PowerScan4 is a compact and portable spectrophotometer designed for rapid and accurate measurements of optical density and absorbance across a wide range of applications. It features a large, easy-to-read display and intuitive user interface, enabling efficient data collection and analysis.
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Lab products found in correlation
9 protocols using powerscan4
1
Triglyceride Quantification in 3T3-L1 Adipocytes
2
Alsterpaullone Activates BZLF1 Reporter
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A luciferase expression reporter plasmid, pTATT‐oriLyt‐Luc, encoding the promoter of the late gene was kindly provided by Eric Johannsen (University of Wisconsin School of Medicine). PSV40‐Rluc (Promega) was used as a control reporter plasmid. HEK293EBV cells were transfected with BZLF1 and reporter plasmids. At 24 hours posttransfection, cells were treated with alsterpaullone. Luminescence in cell lysates was determined by PowerScan 4 (BioTek) at 24 hours after treatment.
3
Cytotoxicity Evaluation of Alkaloids
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3T3-L1 cells were cultured at density of 1.5 × 104 cells/mL in 96-well plates for 16 h. The culture medium was then replaced with fresh medium, treated with alkaloids, and cultured for an additional 24 h. Cell viability was determined using Cell Counting Kit-8 (Dojinbo). The cells were incubated with the reagent for 1 h, and the absorbance of the living cells was measured using a PowerScan4 (Biotek) plate reader at 450 nm.
4
Resazurin-Based Cell Viability Assay
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Firstly, 1 × 105 of NB cells were seeded into 6-well plates. Twenty-four hours after infection, the virus was removed, and the cells were washed for 3 times with PBS, followed by the addition of fresh medium. After 72 hours, the cells were washed for 3 times with PBS. Then 2 ml of serum-free medium with 100 μM resazurin was added. After 2 hours, 100 μl of medium was transferred into 96-wells plate, and the fluorescence was measured by POWERSCAN4 (Bio Tek, λexc = 560 nm, λem = 590 nm).
5
NIR-PIT Cytotoxicity Measurement via Luciferase
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Luciferase activity was used to quantitatively measure the in vitro cytotoxic effects of NIR-PIT with huGPR87ab-IR700. For luciferase activity, 200 μL of 150 μg/mL of D-luciferin-containing media (GoldBio, St Louis, MO, USA) was added to PBS-washed cells 24 h after NIR-PIT. The cells were then analysed on a bioluminescence plate reader (Powerscan4; BioTek, Winooski, VT, USA; cat # BT-S4LFPTAD) at 24 h after NIR-PIT, as previously reported [30 (link),35 (link)]
6
Cytotoxicity Evaluation of NIR-PIT
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The cytotoxic effects of NIR-PIT with NZ-1-IR700 were determined by luciferase activity and flow cytometry with PI staining. For luciferase activity, 150 μg/mL of D-luciferin-containing media (GoldBio, St. Louis, MO, USA) was administered to PBS-washed cells 24 h after NIR-PIT and analyzed with a plate reader for detection of their bioluminescence (Powerscan4, BioTek, Winooski, VT, USA). We evaluated luciferase activity in vitro after NIR-PIT with a few timepoints (1, 6, and 24 h). With this estimation, we decided to evaluate luciferase activity in vitro at 24 h after irradiation.
For the flow cytometric assay, the cells were peeled with pipetting or scraping at 1 h after treatment and washed with PBS. The PI was added to the cell suspension (final 2 μg/mL) and incubated at room temperature for 30 min before flow cytometry. The PI fluorescence was evaluated on ten thousand cells with FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA).
For the flow cytometric assay, the cells were peeled with pipetting or scraping at 1 h after treatment and washed with PBS. The PI was added to the cell suspension (final 2 μg/mL) and incubated at room temperature for 30 min before flow cytometry. The PI fluorescence was evaluated on ten thousand cells with FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA).
7
NIR-PIT Cytotoxicity Evaluation
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The in vitro cytotoxic effects of NIR-PIT with rova-IR700 were determined via luciferase activity as well as PI staining using flow cytometry. For luciferase activity, 200 μL of 150 μg/mL of D-luciferin-containing media (GoldBio, St Louis, MO, USA) was administered to PBS-washed cells 24 h after NIR-PIT; the cells were then analysed on a bioluminescence plate reader (Powerscan4; BioTek, Winooski, VT, USA). We evaluated luciferase activity in vitro 1, 3, 6, and 24 h after NIR light exposure. With this estimation, we decided to evaluate luciferase activity in vitro at 24 h after NIR-PIT. For the flow cytometric assay, cells were washed with PBS twice and dissociated at 1 h after treatment. The cells were detached from the wells by pipetting, and PI was added to the cell suspension (final concentration, 2 μg/mL) and incubated at room temperature for 30 min before performing flow cytometry. PI fluorescence in 10,000 cells was measured using FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA).
8
Evaluating NIR-PIT Efficacy In Vitro
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In vitro, the efficacy of NIR‐PIT was determined via the luciferase activity and the rate of PI‐stained positive rate. For the luciferase assay, the luciferase‐expressing cells were treated with NIR‐PIT using tra‐IR700. After the incubation for 24 h, the luciferase‐expressing cells were washed with PBS, reacted with d ‐luciferin media (150 μg/ml) (GoldBio), and analyzed with a plate scan reader (Powerscan 4; BioTek). For the PI staining assay, the cells were dissociated at 1 h after NIR‐PIT. The cell suspension was incubated with PI (final concentration was set to 2 μg/ml) for 30 min at 25°C. PI‐stained cells were measured by FACS Calibur (Becton Dickinson).
9
Caspase-3/7 Activity Quantification
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Caspase-3/7 activity was quantified by using the Caspase-Glo-3/7 assay system (Promega) according to the manufacturer's instructions. Briefly, 30 µl of Caspase-3/7-Glo assay reagent was added to cells cultured in 96-well plates followed by incubation for 2 h at room temperature. The luminescence was measured using POWER SCAN 4 (BioTek).
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