Compounds used were: Ethanol, chloroform, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sodium chloride, potassium chloride, sodium hydroxide, hydrochloric acid, sulfuric acid, sodium bicarbonate, sodium phosphate dibasic (Merck, Germany), penicillin/streptomycin, trypan blue (Sigma, USA), Roswell Park Memorial Institute medium (RPMI-1640), fetal calf serum (FCS), sodium pyruvate, trypsine, L-glutamine (Gibco, Scotland), dimethyl sulfoxide (DMSO) (Fluka, Italy) and doxorubicin (Farmitalia, Italy).
Trypsine
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Trypsin is a proteolytic enzyme used in cell culture to disrupt cell-cell and cell-extracellular matrix interactions, allowing for the detachment and dissociation of adherent cells. It is commonly used in the passaging or subculturing of adherent cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Trypan blue, by Merck Group (2 mentions)
Sodium phosphate dibasic, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Colchicine, by Merck Group (1 mentions)
Suramin, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
Mtt reagent, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Endothelial cell medium (ecm), by ScienCell (1 mentions)
Collagenase type 5, by Merck Group (1 mentions)
Influx sorter, by BD (1 mentions)
Bovine calf serum (bcs), by Thermo Fisher Scientific (1 mentions)
Vitamin b12, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Dnase, by Merck Group (1 mentions)
9 protocols using trypsine
1
Cytotoxicity Assay of Doxorubicin
2
Mitotic Chromosome Preparation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The incorporation of BrdU into the S phase lasted 6–7 hours. Meanwhile cells were continuously observed by reversed microscope until the number of mitotic round cells peaked. Cells were trypsinysed (trypsine 0.05% + 0.02% EDTA, GIBCO) and harvested in a 15 ml tube with colchicine (final concentration: 0.05 μg/ml, Sigma). After centrifugation, hypotonic treatment was undertaken during 13 min at 37 °C with diluted newborn calf serum (1:5). Intracytoplasmic structures were prefixed with 1 ml of methanol/acetic acid (3:1) at 37 °C. Fixation was finally realised at 4 °C and after centrifugation, 1 ml was let in tubes until spreading. Slides were washed, rubbed and placed in cold water. A few drops from the cell suspension were spread at 10 cm of cold slide and left to dry until staining procedures occurred (adapted from Ladjali et al. 1995 ).
3
Endothelial Cell Culture and Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Endothelial Cell Medium (ECM) supplied with endothelial cell growth supplements (ECGS) was purchased from ScienCell, USA. Cell culture RPMI 1640 medium and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from GIBCO; Trypsine and heat inactivated foetal bovine serum (HIFBS) were obtained from GIBCO, UK. MTT reagents, suramin, aprotinin, 6-aminocarpoic acid, L-glutamine, thrombin, gentamicin were purchased from Sigma-Aldrich, USA.
4
Isolation and Sorting of Mouse Pancreatic Islets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse pancreatic islets were prepared as described previously.45 (link) Briefly, pancreata were digested using type V collagenase (Sigma, ref C9263-1G) for 10 min at 37°C. After digestion, pancreatic islets were separated in a density gradient medium (Histopaque) and purified by handpicking under a binocular magnifier. Pancreatic islets were cultured for 24 h before further processing. For cell sorting experiments, mouse pancreatic islets isolated from RipCre-tdTomato mice were trypsinized for 7 min with 1 mM of trypsine (Gibco). Dissociated pancreatic cells were directly run into an Influx sorter (Becton Dickinson) equipped with a 86 μm nozzle and tuned at a pressure of 24.7 psi and a frequency of 48.25 kHz. Sample fluid pressure was adjusted to reach and event rate of 10 000 events/second. β cells and other pancreatic cells were selected as Tomato + (Tom+, β cells) and Tomato – (Tom-, non-β cells) and sorted in purity mode (phase mask 16/16) directly in RNeasy kit Lysis buffer for further processing.
5
Isolation of Neonatal Rat Ventricular Myocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NRVMs were prepared
from six, 1–3 day old pups, as previously described, with minor
modifications.24 (link),25 (link) The University of Colorado Denver
Animal Care and Use Committee’s regulations were fulfilled
for all animal studies. Using scissors, the ventricles and atria were
separated, and the ventricles were then separated, dissociated in
calcium-free and bicarbonate-free Hanks with Hepes (CBFHH) buffer
containing Heparin (Sigma-Aldrich) (10 U·mL–1), and digested in a CBFHH solution containing 1.12 mg·mL–1 of trypsine (Gibco) and 20 μg·mL–1 of DNAse (Sigma-Aldrich). Two sequential pre-plating steps, on 100
mm dishes in Dulbecco’s modified Eagle’s medium (Gibco),
were used to enrich cardiomyocytes (>90% purity) over non-myocytes.
First, 4.5 g augmented with 5% bovine calf serum (Gibco) and then
2 mg mL–1 vitamin B12 (Sigma-Aldrich) and cultured
unattached cells, predominantly myocytes, were harvested and cultivated
in gelatin-coated dishes before being given various treatments and
further examinations. A minimum of three separate cell isolations
were used to test each experimental condition in triplicate.
from six, 1–3 day old pups, as previously described, with minor
modifications.24 (link),25 (link) The University of Colorado Denver
Animal Care and Use Committee’s regulations were fulfilled
for all animal studies. Using scissors, the ventricles and atria were
separated, and the ventricles were then separated, dissociated in
calcium-free and bicarbonate-free Hanks with Hepes (CBFHH) buffer
containing Heparin (Sigma-Aldrich) (10 U·mL–1), and digested in a CBFHH solution containing 1.12 mg·mL–1 of trypsine (Gibco) and 20 μg·mL–1 of DNAse (Sigma-Aldrich). Two sequential pre-plating steps, on 100
mm dishes in Dulbecco’s modified Eagle’s medium (Gibco),
were used to enrich cardiomyocytes (>90% purity) over non-myocytes.
First, 4.5 g augmented with 5% bovine calf serum (Gibco) and then
2 mg mL–1 vitamin B12 (Sigma-Aldrich) and cultured
unattached cells, predominantly myocytes, were harvested and cultivated
in gelatin-coated dishes before being given various treatments and
further examinations. A minimum of three separate cell isolations
were used to test each experimental condition in triplicate.
6
Cell Viability Assay using Trypan Blue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were trypsinized with a 0.25% solution of trypsine (Gibco; Thermo Fisher Scientific, Inc.) and diluted in 1 ml of supplemented DMEM culture medium. Aliquots of this solution were stained with 0.4% Trypan Blue (Sigma-Aldrich; Merck KGaA) diluted 1:4 in PBS (Gibco) and counted in a Neubauer chamber for assessment of cell viability.
7
Quantitative Real-Time PCR Analysis
8
Steroid and Phthalate Standards Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four steroid standards including E1, E2, E3 and EE2 and BPA were supplied by Sigma-Aldrich (USA). The standards of six phthalates, including DMP, DEP, DBP, BBP, DEHP and DnOP were obtained from Aladdin Reagent Corporation (China). HPLC grade methanol, alkane, acetone, methyl tert-butyl ether (MTBE) and ethyl acetate were supplied by CNW (CNW Technologies GmbH, Germany). Dimethyl sulphoxide (DMSO), sodium hydroxide (NaOH) and a 25% ammonia solution were supplied by Merck (Darmstadt, Germany). Dulbecco's Modified Eagle Medium (DMEM without phenol red (Gibco)), sodium pyruvate (100 mM, sterile-filtered), alpha-Minimal Essential Medium (α-MEM (Gibco)), penicillin-streptomycin, fetal bovine serum (FBS), charcoal-stripped FBS, L-glutamine (200 mM), trypsine (0.5% (Gibco)), phosphate buffered saline (PBS, 1X, pH 7.4) and trypsine without phenol red (10X, 0.5% (Gibco)) were purchased from Life Technologies (United Kingdom). Ultrapure water was obtained using a Milli-Q Advantage A-10 system (Millipore, USA). The physiochemical properties of the investigated steroids, BPA and phthalates are provided in Table S1.
9
HUVEC Culture Optimization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For HUVEC experiments, the following materials were used: bare medium (bM199) consisting of Medium 199 supplemented with penicillin 100 IU ml -1 and streptomycin 100 µg ml -1 (all from Biowhittaker, Verviers, Belgium); Complete medium (cM199) consisting of bare medium (bM199) supplemented with 10% heatinactivate new-born calf serum (Gibco, Grand Island, NY, USA), 10% human serum (from a local blood bank, pooled serum of 10-20 healthy donors, stored at 4°C), 20 mmol litre -1 HEPES (pH 7.3), 2 mmol litre -1 glutamine (both from Biowhittaker), 5 U ml -1 heparin (Leo Pharmaceutical Products, Weesp, The Netherlands), 150 µg ml -1 crude endothelial cell growth factor prepared from bovine hypothalamus; 1% HSA solution (dilution of human serum albumin in bM199; Sanquin CLB, Amsterdam, The Netherlands); Trypsine (Gibco); tissue culture plastics (Costar; Cambridge, MA, USA), and ECIS culture plates with a Z-Theta measurement device and analysis software (Applied BioPhysics).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!