The neutral red uptake assay for cell viability was previously described in detail (80 (link)) and is adapted here. In brief, 1.5 × 104 BHK-21 cells were seeded into 96-well plates and cultured for 24 h in phenol red-free complete growth medium. Cells were then incubated for 8 h or 24 h at 37°C with serial dilutions of 100% DMSO and 50 mM BBC prepared in phenol red-free medium A (minimal essential medium [MEM], 2 mM glutamine, 0.2% bovine serum albumin [BSA], 100 U/ml penicillin, 100 μg/ml streptomycin, and 10 mM HEPES [pH 7.0], medium S (BHK growth medium with 10 mM HEPES [pH 7.0] and 2% FBS instead of 5%), or 2% FBS medium (medium A with 2% FBS instead of BSA). Cells were then incubated for 2 h with 40 μg/ml neutral red solution in phenol red-free complete growth medium, washed, and extracted, and neutral red absorbance was measured from the top on a SpectraMax M5 microplate reader (Molecular Devices) with SoftMax Pro 7.0 software (Molecular Devices). A dose-response curve with a least-squares (ordinary) fit was drawn using Hill function analysis to determine the concentration of BBC causing 50% inhibition of uptake, which was considered the 50% cytotoxic concentration (CC50) of BBC.
Softmax pro 7
Manufactured by Molecular Devices
Sourced in United States
The SoftMax Pro 7 software is a comprehensive data acquisition and analysis platform designed for use with Molecular Devices' microplate readers. The software provides a user-friendly interface for controlling the reader's operations, collecting and processing experimental data, and generating informative reports.
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Lab products found in correlation
Spectramax id5, by Molecular Devices (9 mentions)
Spectramax m5, by Molecular Devices (7 mentions)
Flexstation 3, by Molecular Devices (5 mentions)
Spectramax 190, by Molecular Devices (5 mentions)
Spectramax abs plus, by Molecular Devices (4 mentions)
Spectramax i3x, by Molecular Devices (4 mentions)
Gemini em, by Molecular Devices (4 mentions)
Spectramax i3, by Molecular Devices (4 mentions)
Spectramax m3, by Molecular Devices (3 mentions)
Nano glo hibit lytic detection system, by Promega (3 mentions)
Envision multilabel plate reader, by PerkinElmer (3 mentions)
Envision manager 1, by PerkinElmer (2 mentions)
Sf cell line 4d nucleofector kit, by Lonza (2 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (2 mentions)
Spectramax i3x plate reader, by Molecular Devices (2 mentions)
Oil red o, by Molecular Devices (2 mentions)
Oil red o, by Merck Group (2 mentions)
Spectramax id3, by Molecular Devices (2 mentions)
Fitc d, by Merck Group (2 mentions)
Centrifuge 5430 r, by Eppendorf (2 mentions)
113 protocols using softmax pro 7
1
Neutral Red Uptake Assay for Cell Viability
2
Amyloid-β Aggregation Tracking via Thioflavin T
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The progression of amyloid β aggregation was tracked using thioflavin T (ThT), a fluorescent dye originally used to stain amyloid fibrils in histological samples. ThT (Sigma, product no. T3516) was dissolved in PBS and filtered through a 0.2 μm syringe filter. An aliquot of the Aβ stock solution was diluted to 10 µM in pH 7.4 PBS and incubated at 37 °C with shaking. To observe the inhibitory effect of CLB on Aβ aggregation, 10 µM Aβ was mixed with varying concentrations (0.1, 1, 10, and 20 µM) of CLB. Subsequently, 20 µl of 10 µM ThT was mixed with 20 µl of various concentrations of sonicated fibrils to achieve the desired concentrations of ThT and amyloid. ThT fluorescence was measured at 25 °C using a spectrophotometric plate reader (ELISA reader, Tecan Sunrise, Switzerland) with an excitation filter at 450 nm and an emission filter at 482 nm [21 (link)], the computation of intensity was performed through the utilization of SoftMax Pro 7.0 software (Molecular Devices, CA, USA). After ThT binds to β-sheet aggregate structures, such as amyloid fibrils, its fluorescence emission changes, allowing the monitoring of amyloid β aggregation.
3
Luciferase Reporter Assay for C9orf72 ASOs
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We generated a luciferase construct containing sequences from the human C9orf72 gene (158–900 base pairs) in the 3′-UTR of the renilla luciferase gene in psiCHECK2 vector. An ASO targeting this sequence should decrease renilla luciferase signal without affecting the firefly luciferase signal. We normalized renilla to firefly luciferase signals to compare the relative activity of ASOs versus a non-targeting control (NTC). We delivered ASOs (15 or 30 nM) and the luciferase reporter constructs (20 ng) by transfection with Lipofectamine 2000 into Cos-7 cells. The firefly and renilla luciferase signals were quantified with a plate reader (Molecular Devices Spectramax M5 with SoftMax pro 7.0 software) 48-h post-transfection. We performed three biological replicates per experiment.
4
VEGF Quantification in Corneal Cells
5
Measuring Cellular Reactive Oxygen Levels
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Reactive oxygen species (ROS) were measured using 2′,7′–Dichlorofluorescin diacetate (DCFDA). After 7 days of differentiation, media was removed and cells washed with PBS. Substrates were added (10 mM glucose, 10 mM galactose, or 100 µM palmitate conjugated to 17 µM BSA) and the plate incubated for 24 h (for palmitate assay was run immediately). 20 µM DCFDA was added to cells and incubated at 37 °C, 5% CO2 in the dark for 45 min. DCFDA was removed and cells washed with PBS. PBS supplemented with 10% FBS was added to cells and the plate incubated for 30 min at 37 °C and 5% CO2. Fluorescence was measured (excitation 485 nm; emission 535 nm) using a Molecular Devices SpectraMax M5R and SoftMax Pro 7.0 software. Each measurement was performed in triplicate. Data were normalised to total protein concentration.
6
MTT Assay for Cell Viability
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To measure cell proliferation/viability, 6 replicates of 5000 cells per well were plated in 96 well plates in 100 µL complete media, grown 24 h, and then incubated with 20 µL of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Amresco) at 37 °C for 4 h according to the manufacturer protocol. Supernatants were aspirated and formazan crystals were dissolved in 100 µL DMSO for 2–4 h at 37 °C and then absorbance at 540 nm was measured using a FlexStation3 plate reader using SoftMax Pro 7.0 software (Molecular Devices). Graphed data show average and SEM of biological replicates.
7
Genome Editing and Cell Viability Assay
8
CRBN-Dependent Cytotoxicity Assay
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HEK293T cells or HEK293T cells with CRBN+/+ or CRBN−/− (approximately 0.25 × 106 cells) were nucleofected with 400 ng of LSD-Cas9 plasmid or 400 ng of Cas9-P2A-CSD-AcrIIA4 plasmid along with 40 ng of GAPDH-targeting gRNA plasmid, and 40 pmol of HiBiT ssODN using the SF Cell Line 4D-Nucleofector kit (Lonza) following the pulse program of CM-130. Approximately 15,000 cells were seeded in a well in a 96-well plate in 100 µL of DMEM media. Cells were incubated at 37°C for 48 h (for CRBN−/− and CRBN+/+) or 72 h (HEK293T, time-dependent study) in the presence of the indicated amount of pomalidomide. Next, the cell viability was measured using the PrestoBlue Cell Viability Reagent (Thermo) with SpectraMax M5 and SoftMax Pro 7.0 (Molecular Devices). Next, HiBiT detection was performed using the Nano-Glo HiBiT Lytic Detection System (Promega) according to the manufacturer’s protocol. The luminescence signal was recorded by an EnVision Multilabel Plate Reader with EnVision Manager 1.13 (PerkinElmer). The resulting luminescence signals were normalized with PrestoBlue-based cell viability.
9
Quantifying Serum Antibody and Cecal IgA
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For serum antibody responses, mice were immunized s.c. with a total of 100 μg NP-CGG (Biosearch Technologies Inc.) in alum (Sigma-Aldrich) divided among four sites. Serum was prepared from blood collected from the retro-orbital space 14 d later. For detection of cecal IgA, cecal matter was suspended in PBS at 50 mg/ml and centrifuged at 400 g for 5 min, supernatant was collected and centrifuged again at 8,000 g for 10 min, and supernatants were used for detection. Microtiter plates were coated overnight at 4°C with 10 μg/ml NP2 or NP30-BSA (Biosearch Technologies Inc.) or 2 μg/ml anti-IgA (C10-3; BD) diluted in carbonate buffer, pH 9.6. Washing was with PBS with 0.1% Tween-20 (PBST), blocking was with PBST/5% BSA, and serum or cecal supernatants were diluted in PBST/1% BSA. Serum or cecal dilutions were incubated in the coated wells for 1 h, and bound antibodies were detected using anti-IgG1 or anti-IgA conjugated to HRP (Southern Biotech) developed with 3,3′,5,5′ tetramethyl benzidine (BioLegend). Reactions were stopped after 5 min with 2 N H2SO4. Absorbance was measured at 450 nm in a SpectraMax M3 microplate reader using SoftMax pro 7.0 (Molecular Devices). Purified mouse IgA (Southern Biotech) served as the standard for cecal samples.
10
Quantifying Protein Levels in Hemolymph and Oocytes
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The total amount of protein levels in the hemolymph and oocytes were measured using the Lowry (Folin) method, with 1–7 µg of BSA serving as the standard control (Lowry et al., 1951 (link)). Measurements were performed in an E-MAX Plus microplate reader (Molecular Devices) using SoftMax Pro 7.0 as software. For each biological replicate, hemolymph samples were obtained from 1 individual. For oocyte homogenates, a pool of 2 choriogenic oocytes was obtained from 1 individual per biological replicate.
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