Typically, HUVECs were cultured in a humidified incubator (5% CO2, 37 °C). For cell adhesion assay, HUVECs (1 × 105 per well) were seeded on the 24-well plates containing HF or PNS-HF scaffolds (scaffold size: 20 mm × 20 mm × 2 mm, PNS content: 25 μg/ml). The cells and scaffolds were stained using a LIVE/DEAD Cell Imaging kit after 24 h of culture. For proliferation assay, the HUVECs were incubated with HF or PNS-HF scaffolds for 5 days, and CCK8 assay was conducted every 2 days. For a typical CCK8 assay, the culture medium was replaced with fresh medium containing the CCK8 kit (10% v/v). The medium was pipetted after incubation of 2 h, and the absorbance at 450 nm was measured via a microplate reader (Epoch, BIO-TEK).
Bio tek
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BIO-TEK is a line of laboratory equipment manufactured by Agilent Technologies. The core function of BIO-TEK products is to facilitate various scientific experiments and analyses in research and diagnostic settings.
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Lab products found in correlation
Celltox green dye, by Promega (1 mentions)
Prism 8, by GraphPad (1 mentions)
Sod a001 1, by Nanjing Jiancheng (1 mentions)
β actin c4, by Santa Cruz Biotechnology (1 mentions)
Histone 1 ae 4, by Santa Cruz Biotechnology (1 mentions)
Image lab 5.2 analysis software, by Bio-Rad (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Cck 8 assay, by Keygen Biotech (1 mentions)
Synergy h1 multi mode microplate reader, by Agilent Technologies (1 mentions)
Rneasy lipid tissue, by Qiagen (1 mentions)
Take3 plate, by Agilent Technologies (1 mentions)
7500ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Cytation 3, by Agilent Technologies (1 mentions)
Propidium iodide, by Coolaber (1 mentions)
Mtt assay, by Meilun (1 mentions)
Multi detection microplate reader, by Agilent Technologies (1 mentions)
Human leptin elisa kit, by Boster Bio (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
31 protocols using bio tek
1
Cell Adhesion and Proliferation on Scaffolds
2
In Vitro Cytotoxicity Evaluation of Nano-Bubbles
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PC9GR cells were uniformly planted in 96-well plates with a density of 5000 cells per well, given 100 µL RPMI-1640 medium containing 10% fetal bovine serum, and then cultured at 37 °C with 5% CO2 overnight. The cytotoxicity assays were divided into two groups: group I, the original medium was changed to a fresh medium containing PBS (Control), blank NBs, SCR siRNA-NBs or Naked SCR siRNA (at siRNA-NBs volume 10 µL/well or siRNA 6 pmol/well) and cultured with PC9GR cells for 12 h. PC9GR was replaced with fresh medium and cultured for 48 h; group II, the original medium was changed to a fresh medium containing PBS (Control), different concentrations of blank NBs, or SCR siRNA-NBs (at 1.0 × 106, 5.0 × 106, 1.0 × 107, 5.0 × 107, 1.0 × 108, and 5.0 × 108 bubbles/mL) and cultured with PC9GR cells for 12 h. After the cells were rinsed with PBS twice, 100 µL of new culture medium was replaced in each well, and 10 µL Cell Counting Kit-8 (CCK-8) was added, then the cells were put back into the incubator for further incubation for 3 h. Finally, the absorbance was measured at 450 nm with an enzyme plate meter (Bio Tek, Proton Instrument Co., Ltd., USA). The above experiment process is repeated three times.
3
Permeability Quantification in BBB Chip
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Culture medium containing 100 μg mL−1 of dextran (3 kDa) and 20 μg mL−1 of lucifer (0.5 kDa) tracers were dosed through the vascular channels for 24 h, and concentration of the dextran and lucifer tracers in the outlet samples from both vascular and brain channels was determined by using BioTek (BioTek Instruments, Inc., Winooski, VT, USA). Then, the following Eq. (1 ) was used to calculate Papp:
Here, SA is the surface area of sections of the channels that overlap (0.17cm2), QD and QR are the fluid flow rates in the dosing and receiving channels respectively, in units of cm3/s, CD,O, and CR,O are the recovered concentrations in the dosing and receiving channels respectively, in any consistent units. IgG permeability was also evaluated after dosing the vascular channel and measuring the IgG content on the brain channel. Detection and quantitation of serum immunoglobulin G (IgG1; Abcam) was performed using the ELISA kit (Abcam), after 24 h of perfusion.
Here, SA is the surface area of sections of the channels that overlap (0.17cm2), QD and QR are the fluid flow rates in the dosing and receiving channels respectively, in units of cm3/s, CD,O, and CR,O are the recovered concentrations in the dosing and receiving channels respectively, in any consistent units. IgG permeability was also evaluated after dosing the vascular channel and measuring the IgG content on the brain channel. Detection and quantitation of serum immunoglobulin G (IgG1; Abcam) was performed using the ELISA kit (Abcam), after 24 h of perfusion.
4
Cytotoxicity Assay for Drug Screening
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2500 ONS-76 cells were seeded in 100 µl per well of complete RPMI medium in 96-well low adhesion plate (Greiner bio-one, 650970, Germany) for 24 h to form spheroids. After spheroid formation, 50 µl of medium per well were removed and replaced by 50 µl fresh medium containing increasing concentrations of drugs, complemented with 1:500 of CellTox™ Green Dye (G8742, Promega) as described by the manufacturer. The fluorescence levels representing the nonviable cell population were measured after 24, 48 and 72 h using a Cytation 3 imaging reader BioTek® (λex = 485 nm, λem = 520 nm). The cytotoxicity curve was generated with GraphPad Prism 8 software as log (inhibitor) vs. response − Variable slope (four parameters) with 100% as the effect observed with the highest concentration of dasatinib (80 µM).
5
Hemin Detoxification Bioassay Protocol
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Bioassay was carried out by the inhibition test of heme detoxification (ITHD) method (22 (link)). The test was carried out at hemin concentrations (30, 60, 120 μM), pHs (3.6, 4, 4.4, 4.8, and 5) at 37 °C, 60 °C three times (4, 24, 48 h). Bioassay procedure was as follows, hemin chloride was dissolved in dimethyl sulfoxide (DMSO) diluted to 60 μg/mL by sodium acetate buffer (1 M, pH = 4.5), instead of β-hematin, hemin was employed in acidic condition (acetate buffer), Tween® 20 was diluted to 0.012 g/L with distilled water and synthesized compounds were dissolved by DMSO. Hemin, samples, and Tween® 20 were distributed in each well of a 96-well plate with a ratio of 9:9:2, respectively, in triplicate. The final concentration of all the materials in each well was 200 μg/mL (22 (link)).
As a control, these samples were prepared in triplicate under the same condition in lack of hemin. These controls allowed being prevented interference absorption of those remains from matrix samples. Negative control samples were dissolved in DMSO without hemin. Chloroquine phosphate was used as a positive control. The plates were incubated at 60 °C for 24 h. Finally, the materials in plates were mixed with a micropipette and then adsorption of the solutions was read at 405 nm by ELISA reader (Epoch, BioTeK, USA).
As a control, these samples were prepared in triplicate under the same condition in lack of hemin. These controls allowed being prevented interference absorption of those remains from matrix samples. Negative control samples were dissolved in DMSO without hemin. Chloroquine phosphate was used as a positive control. The plates were incubated at 60 °C for 24 h. Finally, the materials in plates were mixed with a micropipette and then adsorption of the solutions was read at 405 nm by ELISA reader (Epoch, BioTeK, USA).
6
Serum and Kidney Biochemical Analysis
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All blood samples were allowed to clot at room temperature and centrifuged at 2,000 g for 10 min to harvest serum. Serum biochemical parameters of BUN, and Scr and CK levels were measured (n = 12 per group) using the commercially available kits, BUN (995–17711), Scr (991–32593), and CK (994–64291), all from Wako Pure Chemical Industry, Japan.
The kidneys were excised, then washed in ice-cold saline and homogenized in 0.1 M Tris–HCl buffer (pH 7.4). The homogenate were first centrifuged at 10,000 g for 15 min and the supernatants were then centrifuged at 100,000 g for 1 h. The resulting supernatant (cytosolic fraction) was used for the determination of enzymatic activities and lipid peroxidation. Kidney biochemical parameters of SOD, MDA and GSH-Px were measured spectrophotometrically (Eon, BioTeK, USA) using the commercially available kits, SOD (A001-1), MDA (A003-1), and GSH-Px (A005), all from Jiancheng Bioengineering Institute, Nanjing, China.
The kidneys were excised, then washed in ice-cold saline and homogenized in 0.1 M Tris–HCl buffer (pH 7.4). The homogenate were first centrifuged at 10,000 g for 15 min and the supernatants were then centrifuged at 100,000 g for 1 h. The resulting supernatant (cytosolic fraction) was used for the determination of enzymatic activities and lipid peroxidation. Kidney biochemical parameters of SOD, MDA and GSH-Px were measured spectrophotometrically (Eon, BioTeK, USA) using the commercially available kits, SOD (A001-1), MDA (A003-1), and GSH-Px (A005), all from Jiancheng Bioengineering Institute, Nanjing, China.
7
Quantifying STAT3, NF-κB, and Bcl-2 Protein Levels
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Western blot analysis and a direct enzyme‐linked immunosorbent assay (ELISA) were performed, as previously described7 , 13 , 16 , 31 and in Supplementary Material , to monitor the successful knockdown of STAT3 expression, and to determine the effect of STAT3 knockdown or its pharmacologic inhibition on BA‐induced STAT3, NF‐κB and Bcl‐2 protein levels. We used primary antibodies for p‐STAT3 (Tyr 705) (clone B‐7), STAT3 (clone F‐2), p‐NF‐κB (p65 Antibody 27. Ser 536), bcl2 (Clone N‐19), Histone 1 (AE‐4) and β‐actin (C4) (Santa Cruz Biotechnology Inc.). Protein levels obtained by Western blot analysis were quantified by the Gel imaging system (Bio‐Rad) in each nuclear or cytoplasmic cellular compartment (Image Lab 5.2 analysis software, Bio‐Rad). Protein levels obtained by ELISA were quantified by Gen5™ software reading the absorbance values using a microplate reader (Sunergy1, BIOTEK; Gen5™ software; BIOTEK Instruments Inc.). Assays were carried out according to the manufacturer's instructions and performed in triplicates and repeated two times, independently.
8
Cell Proliferation Assay via CCK8
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The cells were seeded in 96 well plate at a density of 2 × 103 cells per well. Cell proliferation were detected by CCK8 assay according to manufacturer’s instructions (KeyGENBiotech, Nanjing, China). In short, at different time point, 10 μl of CCK8 reagent was added into each well and the cells were incubated for 2 h at 37°C. Then, the absorbance value was measured at 450 nm by using the microplate reader (Epoch, BIO-TEK). The relative viability of cells was calculated as a percentage using the formula: (mean OD450 of treated cells/mean OD450 of control cells) × 100%.
9
Quantifying Pyoverdine Secretion in P. aeruginosa
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The PA01 strain of P. aeruginosa was used for quantifying pyoverdine secretion. P. aeruginosa was incubated without any drug (growth control) and also in media supplemented with sub-MIC doses of gentamicin and compounds at 37 °C for 48 h. The cells in each culture media was harvested by centrifuging for 40 min at 4000 rpm. For pyoverdine quantification, 100 μL of the cell-free supernatant was pipetted into a microtiter plate. Fluorescence measurements were carried out at 405 nm for excitation and 465 nm for emission on a BioTeK® Synergy H1 Multimode Microplate Reader (Germany) [15 (link), 25 , 26 (link)]. Percentage inhibition was computed relative to the control by applying the expression in [1 ].
10
Quantitative Gene Expression Analysis in Adipose Tissue
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Total RNA was extracted from 3T3 cells using TRIzol® (Ambion, Austin, TX) and from frozen adipose tissue by RNeasy® Lipid Tissue (Qiagen), as per instructions provided by the manufacturers. RNA was determined by measuring the absorbance at 260 nm (A260) with a Biotek™ plate reader and the Take3™ plate (Biotek, Winooski, VT), and assessed by the A260/A280 ratio. cDNA was synthesized from total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), after real-time PCR was performed using TaqMan® Fast Universal Master Mix (2x), on a 7500 HT Fast Real-Time PCR System (Applied Biosystems). Specific TaqMan Gene Expression Assays probes for mouse HO-1, PGC1α, UCP1, COX-IV (cytochrome c oxidase subunit-IV), adiponectin, TNFα, IL-6, Mfn1, Mfn2, Drp1, Fis1, OPA1, aP2, SIRT3, C/EBPα, and GAPDH were used as previously described [33 (link)].
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