Following the habituation phase, all animals were deeply anesthetized with an intraperitoneal injection of pentobarbital (Morbital, Biowet, Poland; 2 mL/kg body weight), and after cessation of breathing, immediately perfused transcardially with saline (0.9%) followed by 4% paraformaldehyde (pH 7.4; 1040051000, Sigma Aldrich, Taufkirchen, Germany) in phosphate-buffered saline (PBS; P5493, Sigma Aldrich, Taufkirchen, Germany). After perfusion, brains were carefully dissected from the skulls and post-fixed by immersion in the same fixative for 24 h, washed twice in 0.1 M phosphate buffer (pH = 7.4, 4 °C), and then stored for 3–5 days in graded solutions (19% and 30%) of sucrose (S0389, Sigma Aldrich, Saint Louis, MO, USA) in 1×PBS at 4 °C until full saturation. Finally, the brains were frozen and then coronally sectioned at a thickness of 10 μm using a cryostat (HM525 Zeiss, Germany). The sections were mounted on object slides and stored at −80 °C until further processing.
P5493
Manufactured by Merck Group
Sourced in United States, Germany
P5493 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of the product is to provide a reliable and consistent performance for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Hm525, by Zeiss (5 mentions)
S0389, by Merck Group (3 mentions)
P6148, by Merck Group (3 mentions)
C0775, by Merck Group (2 mentions)
H1758, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Hy d1056, by MedChemExpress (1 mentions)
D2650, by Merck Group (1 mentions)
Caco 2 cells htb 37, by American Type Culture Collection (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
F8687, by Merck Group (1 mentions)
Scr103, by Merck Group (1 mentions)
L2630, by Merck Group (1 mentions)
Morbital, by Biowet (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Giemsa, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Transwell chamber, by Corning (1 mentions)
Trypsin, by Merck Group (1 mentions)
P7000, by Merck Group (1 mentions)
22 protocols using p5493
1
Brain Tissue Fixation and Sectioning
2
Caco-2 Cell Viability Assay with LPS and Glycyrrhizic Acid
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Human colorectal adenocarcinoma epithelial cells, Caco-2 cells (HTB-37), were purchased from American Type Culture Collection (ATCC, Manassas, VA), and cultured in ATCC-formulated Eagle’s Minimum Essential Media (30-2003, ATCC, Manassas, VA) supplemented with 20% foetal bovine serum (FBS, F2442, Sigma-Aldrich, St. Louis, MO) at 37 °C with 5% CO2. GP (HY-N6881, C41H70O12, purity: ≥90.0%, CAS no. 80325-22-0, MedChemExpress, Monmouth Junction, NJ) were solubilized in dimethyl sulphoxide (D2650, DMSO, Sigma-Aldrich, St. Louis, MO), and diluted into the gradient concentrations of 0, 50, 100, 150 or 200 μM for the use in drug treatment. Notably, DMSO was set at the 0.1% final concentration in in vitro experiments. Also, gradient GP were applied in the measurement of cell viability. Then, Caco-2 cells were assigned into four groups (control group, LPS group, LPS + GP150 group and LPS + GP200 group). Caco-2 cells in the LPS + GP150 group or the LPS + GP200 group were treated with 150 μM or 200 μM of GP at 37 °C for 24 h with 5% CO2. After being washed with phosphate-buffered saline (PBS, P5493, Sigma-Aldrich, St. Louis, MO), cells were exposed to 10 μg/mL LPS (HY-D1056, LPS, MedChemExpress, Monmouth Junction, NJ) at 37 °C for 24 h with 5% CO2 (He et al. 2020 (link)). Cells in the LPS group only underwent 10 μg/mL LPS exposure for 24 h, and cells in the Control group remained untreated.
3
Isolation and Culture of HPDLSCs
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All the cell culture procedures were authorized by the ethics committee of Xi'an Jiaotong University Stomatology Hospital. HPDLSCs were isolated and cultured as previously described 15 (link), 16 (link). In brief, healthy third molars were obtained after obtaining informed consent from female patients aged 18-24. The obtained third molars were washed thoroughly with phosphate buffered saline (PBS) (P5493, Sigma-Aldrich, USA). Then, the periodontal ligament tissue in the middle third of the third molar root was scraped and treated with 3 mg/mL collagenase type I (SCR103, Sigma-Aldrich, Missouri, USA) at 37°C for 1 h. Cell suspensions were incubated at 37°C in 5% CO2 using α-MEM (32561102, ThermoFisher, Massachusetts, USA) containing 20% fetal bovine serum (F8687, Sigma-Aldrich, USA). Cells were cultured in standard medium (α-MEM with 10% fetal bovine serum) and passaged at approximately 80%-90% confluence, and the cultured third passage cells were used for subsequent experimental studies.
4
Immune Response of Bats to LPS
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Individuals were randomly assigned to a control (n=6, hereafter ‘control bats’) and a treatment group (n=20, challenged bats). Challenged bats were injected subcutaneously with LPS from Escherichia coli O111:B4 (L2630, Sigma Aldrich, MO, USA) at a concentration of 2mg/kg bodyweight (0.577 ± 0.144mg) diluted in sterile phosphate-buffered saline (PBS, P5493, Sigma-Aldrich, MO, USA) (28 (link)). The experiment (i.e., injection of LPS or PBS) was conducted in two rounds, each consisting of 10 challenged bats and three control bats (total of 13 bats per round). GM and blood samples and body weight measurements were taken for each bat at three time points - pre-injection, 24h and 48h post-injection (Data Sheet S1
5
Sham Treatments in Rat Model
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The rats received two intraperitoneal injections of 0.9% saline at two-hour intervals, one systemic injection (through the caudal vein) of 0.5 ml phosphate-buffered saline (PBS) (P5493, Sigma, USA) without MSCs and 1 ml saline orally with gastric gavage once daily for 30 days.
6
Brain Tissue Fixation and Sectioning
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Following the habituation phase, all animals were deeply anesthetized with an intraperitoneal injection of pentobarbital (Morbital, Biowet, Poland; 2 mL/kg body weight), and after cessation of breathing, immediately perfused transcardially with saline (0.9%) followed by 4% paraformaldehyde (pH 7.4; 1040051000, Sigma Aldrich, Taufkirchen, Germany) in phosphate-buffered saline (PBS; P5493, Sigma Aldrich, Taufkirchen, Germany). After perfusion, brains were carefully dissected from the skulls and post-fixed by immersion in the same fixative for 24 h, washed twice in 0.1 M phosphate buffer (pH = 7.4, 4 °C), and then stored for 3–5 days in graded solutions (19% and 30%) of sucrose (S0389, Sigma Aldrich, Saint Louis, MO, USA) in 1×PBS at 4 °C until full saturation. Finally, the brains were frozen and then coronally sectioned at a thickness of 10 μm using a cryostat (HM525 Zeiss, Germany). The sections were mounted on object slides and stored at −80 °C until further processing.
7
Perfusion and Cryoprotection of Rat Brains
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After the habituation phase, all SHRs and WKYs were randomly allocated into seven age groups in accordance with the study plan, with all animals from each group later given the same tissue processing. Briefly, the rats were acutely anesthetized with an intraperitoneal inoculation of Morbital (Biowet, Puławy, Poland; 2 ml/kg, 133.3 mg/ml of pentobarbital sodium salt and 26.7 mg/ml of pentobarbital). Following cessation of breathing, animals were immediately perfused transcardially with saline (0.9%) followed by 4% paraformaldehyde (PFA; pH 7.4 (1040051000; Merck, Darmstadt, Germany), which was dissolved in phosphate-buffered saline (PBS) (P5493; Sigma-Aldrich, Schnelldorf, Germany). After perfusion, the whole brain was conscientiously removed from the skull. In the next step, brains were post-fixed by immersion in 4% PFA for 24 h and then rinsed three times in 0.1 M phosphate buffer (pH 7.4, 4°C). Thereafter, all brains were cryoprotected in series (10%, 20%, and 30%) with sucrose (363-117720907; Alchem, Wrocław, Poland) in 1× PBS at 4°C until they sunk (3–5 days). Conclusively, the brains were frozen as blocks and were then coronally cut at a thickness of 10 μm using a cryostat (HM525; Zeiss, Jena, Germany). The tissue sections were placed on glass slides and stored at −80°C until further investigation.
8
Flow Cytometry Cell Labeling and Analysis
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Cells were centrifuged at 400 g for 5 min, resuspended in 4 °C PBS (Sigma-Aldrich P5493), plated in multi-well round-bottom plates and immunolabeled for FACS analysis. Desired antibody mixtures were added to 1% BSA/PBS and incubated for 20 min at 4 °C. Flow cytometry evaluation was performed using a BD Biosciences LSR Fortessa flow cytometer, and data were analyzed using FlowJo 10.
9
Transwell Assay for Cell Migration and Invasion
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Transwell chambers (3428, Corning, Corning, NY, USA) were used to assess the migratory and invasive abilities of SiHa cells and ME180 cells after transfection with FAM201A/FLOT1 overexpression plasmids or miR-1271-5p M alone or in combination. The upper chamber was precoated by Matrigel (dilution: 1 : 3; 356234, Corning, USA) for cell invasion assay, while that without Matrigel was used for cell migration assay. The cells were cultured to prepare a cell suspension at a concentration of 5 × 105 cells/ml. Then, 100 μL of the cell suspension was poured into the upper chamber, and 600 μL of DMEM containing 10% FBS was added to the lower chamber. The whole Transwell set was incubated at 37°C for 24 h. Later, the lower chamber was washed twice with phosphate-buffered saline (P5493, Sigma-Aldrich, USA), fixed with 4% paraformaldehyde (P6148 Sigma-Aldrich, USA) and stained with 800 μL Giemsa (10092013, ThermoFisher, USA). After removing nonmigratory or noninvading cells, the remaining cells were observed under × 200 magnification using an inverted microscope (IX71; Olympus, Tokyo, Japan). Cells in five randomly selected fields were counted using ImageJ software and cell migration and invasion rates were calculated.
10
Clonogenic Assay for LINC00665 and miR-339-3p
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After transfection with siLINC00665, miR‐339‐3p mimic, or miR‐339‐3p inhibitor or co‐transfection with siLINC00665 and miR‐339‐3p inhibitor, A375 cells and A2058 cells were detached by Trypsin (9002‐07‐7, Sigma‐Aldrich) and plated at 1 × 105 cells/per well in 6‐well plates, respectively. Then, the cells were allowed to clone at 37°C for 2 weeks, after which colonies were washed with phosphate‐buffered saline (PBS; P5493, Sigma‐Aldrich), fixed in 4% paraformaldehyde (P6148, Sigma‐Aldrich) for 15 min, and stained with 0.1% crystal violet (C0775, Sigma‐Aldrich) for 10 min. The number of colonies with diameters of longer than 1.5 mm in six randomly selected fields was counted under microscope (IX71; Olympus).
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