HIF-1α transcriptional factor activity was quantify by an ELISA-based kit (47096, TransAM Kit, Vinci-Biochem, Firenze, Italy) following the manufacturer’s instructions. Briefly, 8 µg of nuclear extracts obtained by using the Nuclear Extract Kit (40010, Vinci-Biochem) were loaded on the coated plate and analyzed by reading at 450 nm with Gen5 Microplate Collection & Analysis Software Data (BioTek Instruments, Inc.®, Winooski, VT, USA). The data were expressed as the ratio between HIF-1α protein content and total nuclear extracts (absorbance) or in terms of FOI compared to positive control.
Gen5 microplate collection analysis software data
Manufactured by Agilent Technologies
Sourced in United States
Gen5 Microplate Collection & Analysis Software is a versatile software suite designed for automated data collection and analysis from microplate-based experiments. It provides a comprehensive platform for managing and interpreting results from a variety of microplate-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Transam kit, by Active Motif (1 mentions)
Nuclear extract kit, by Active Motif (1 mentions)
Elx808 absorbance microplate reader, by Agilent Technologies (1 mentions)
Synergy 2 multi detection microplate reader, by Agilent Technologies (1 mentions)
Phosstop, by Roche (1 mentions)
Complete mini, by Roche (1 mentions)
10 protocols using gen5 microplate collection analysis software data
1
Quantifying HIF-1α Transcriptional Activity
2
Quantifying HIF-1α Transcriptional Activity
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An ELISA-based kit (TransAM Kit, Vinci-Biochem, Italy) was used to detect and quantify HIF-1α transcriptional factor activity following the manufacturer's instructions. Briefly, nuclear extracts were firstly prepared using the Nuclear Extract Kit (Vinci-Biochem) and 8µg of the samples were added to the coated plate and analysed at 450nm with Gen5 Microplate Collection & Analysis Software Data (BioTek Instruments, Inc.®). Data were expressed as HIF-1α protein content in the total nuclear extract (Absorbance).
3
Quantifying HIF-1α Transcriptional Activity
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An ELISA-based kit (TransAM Kit, Vinci-Biochem, Italy) was used to detect and quantify HIF-1α transcriptional factor activity following manufacturer's instructions. Briefly, nuclear extracts were firstly prepared using the Nuclear Extract Kit (Vinci-Biochem) and 8 μg of the samples were added to the coated plate and analysed at 450 nm with Gen5 Microplate Collection & Analysis Software Data (BioTek Instruments, Inc.®). Data were expressed as HIF-1α protein content in total nuclear extract (Absorbance).
4
Curcumin Modulates HIF-1α Activity
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K562 and LAMA84 cells treated with 20 μM of curcumin were harvested after 24 h of incubation. K562 cells were also transfected or not with miR-22 inhibitor. Nuclear extracts were prepared by using the Nuclear Extract Kit from Activemotif (#40010 - Activemotif, Carlsbad, CA, USA) according to the manufacturer’s instructions. The transcriptional activity of HIF-1α was assayed by an ELISA-based kit (#47096 - TransAM Kit, Activemotif, Carlsbad, CA, USA) according to the manufacturer’s instructions. Nuclear extract samples were added to the coated plate and analyzed at 450 nm with Gen5 Microplate Collection & Analysis Software Data (BioTek Instruments, Inc.®). HIF-1α activity was expressed as Absorbance value.
5
HIF-1α Transcriptional Activity Assay
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An ELISA-based kit (TransAM Kit, Vinci-Biochem, Vinci (Firenze), Italy) was used to detect and quantify HIF-1α transcriptional factor activity following the manufacturer’s instructions. Briefly, nuclear extracts were first prepared using the Nuclear Extract Kit (Vinci-Biochem). A total of 8–10 µg of the samples were added to the coated plate and analyzed at 450 nm with Gen5 Microplate Collection & Analysis Software Data (BioTek Instruments, Bad Friedrichshall, Germany). Data were expressed as HIF-1α protein content in total nuclear extract (Absorbance) or in terms of FOI compared to control cells.
6
Lupin-specific IgG1 Antibody Detection
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The detection of lupin-specific IgG1 antibodies was performed according to Vinje et al. [23 (link)]. In brief; microtiter plates with 96 wells were coated with lupin extract (5 μg/ml), incubated for 1 h at room temperature (RT) and then overnight at 4 °C. After washing (Tris/Tween), blocking (Tris/Tween with 1 % bovine serum albumin) for 1 h, and subsequent washing, plates were incubated for 2 h at RT with lupin-specific IgG1 standard serum in fourfold dilutions for standard curve generation, negative control serum and the sera collected at day 34 (diluted 1:100 in BSA/Tris/Tween). After washing, peroxidase-labelled rat anti-mouse IgG1 antibody was added to each well. Following 2 h incubation at RT, and washing, plates were incubated for 15 min with peroxidase substrate. Plates were read at 405 nm in a BioTek Elx808 Absorbance Microplate reader using Gen5™ Microplate Data Collection & Analysis Software (BioTek® Instruments, Inc., Winooski, Vermont, USA).
7
Amniotic Fluid Cytokine Profiling
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Free‐leaking amniotic fluid was obtained vaginally with a sterile speculum. Samples were collected every second day, centrifuged at 3000 rpm for 5 min at 4℃, and stored at −80℃. Samples containing obvious mucus, blood or too little volume were excluded from the study. Thus, amniotic fluid samples from 156 participants, who delivered within 48 h following amniotic fluid collection, were included into the final analysis.
Immunological assays of amniotic fluid samples were performed using the enzyme‐linked immunosorbent assay (ELISA) for human sTLR 2 and 4 (USCN Life Science & Technology Company), according to the manufacturer's instructions. The samples of amniotic fluid were not diluted for the determination of sTLR2 and sTLR4 concentrations. The concentrations of cytokines were calculated according to standard curves using a special program for the evaluation of ELISA results (Gen5 Microplate Data Collection & Analysis Software; BioTek Instruments).
Immunological assays of amniotic fluid samples were performed using the enzyme‐linked immunosorbent assay (ELISA) for human sTLR 2 and 4 (USCN Life Science & Technology Company), according to the manufacturer's instructions. The samples of amniotic fluid were not diluted for the determination of sTLR2 and sTLR4 concentrations. The concentrations of cytokines were calculated according to standard curves using a special program for the evaluation of ELISA results (Gen5 Microplate Data Collection & Analysis Software; BioTek Instruments).
8
Quantifying Total Protein from Rat Tissue Lysates
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Frozen tissues from control and treated rats were homogenized using PowerGen 500 homogenizer (Fisher Scientific, Rockford, IL) in ice-cold lysis buffer containing 25% glycerol, 62.5 mMTris-HCl, 1xprotease inhibitors (Roche, Complete mini) and phosphotase inhibitors (Roche, PhosSTOP). 10% SDS was added to the samples, vortexed and boiled for 4 min. The extracts were centrifuged at 10,000 rpm for 15 min at 4°C, and supernatants with the total protein were collected. Protein concentration in each sample was detected using BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL). Bovine serum albumin (BSA) was used to generate the standard curve. Each protein sample was diluted 1∶20 with 1% SDS. All standards and samples were run in duplicate. The absorbance was measured at 562 nm on the Synergy 2 Multi-Detection Microplate Reader (BioTek Instruments, Winooski, VT) and data analysis was performed using Gen5 Microplate Data Collection & Analysis Software (BioTek Instruments, Winooski, VT).
9
Quantifying Serum BAFF Levels
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Serum samples were analyzed using commercial BAFF, Soluble (human) ELISA Kit (hypersensitive) (AdipoGen, Switzerland). From collected blood samples, serum was separated and stored at −80°C until analysis. Serum dilutions and enzyme-linked immunoassay was carried out in the strict accordance with the manufacturer's instructions and sets recommendations. The results were evaluated by spectrophotometer (BioTek Instruments, USA). The concentrations of analytes in ELISA assays were quantified using standard curves. A regression analysis was performed to derive an equation that was then used to predict the concentration of the unknown samples with Gen5 Microplate Data Collection & Analysis Software (BioTek Instruments, USA). The results are shown in Table 1 .
10
Microplate Assay for Essential Oil IC50
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The results were processed using BioTek Gen5 Microplate Data Collection & Analysis Software (BioTek, Instruments, Inc., Winooski, VT, USA) which was subsequently analyzed using Microsoft Excel. GraphPad Prism version 8 was used to calculate the IC50 value of essential oil. The results are presented as the mean ± standard deviation of the mean of triplicate data.
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