A purification system (Milli-Q, Merck Millipore, Darmstadt, Germany) was used to provide purified water (18.2 MΩ cm). Nitric acid (HNO3) Rotipuran p. a. ≥ 65% (Carl Roth, Karlsruhe, Germany) was sub-boiled with an MLS duoPUR (MLS, Leutkirch, Germany) prior to its use for the preparation of samples. For internal standards and the preparation of calibration standards, we used ICP Single-Element Standards Certipur (Merck Millipore, Darmstadt, Germany) and Single Element Standards for ICP (Carl Roth, Karlsruhe, Germany). Fifteen and fifty mL Cellstar polypropylene tubes (Greiner Bio-One International GmbH, Kremsmünster, Austria) were used for the preparation of all solutions.
Cellstar polypropylene tube
Manufactured by Greiner
Sourced in Austria
Cellstar polypropylene tubes are laboratory equipment designed for general sample storage and handling applications. They are made of high-quality polypropylene material, providing a durable and reliable solution for various laboratory tasks.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Icp single element standards certipur, by Merck Group (2 mentions)
Single element standards for icp, by Carl Roth (2 mentions)
Milli q, by Merck Group (2 mentions)
Ktaprime plus liquid chromatography system, by GE Healthcare (1 mentions)
Kobe 1 agar, by Carl Roth (1 mentions)
Petri dishes, by Sarstedt (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Trypan blue, by Avantor (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution 10, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline, by Biowest (1 mentions)
Versene solution, by Thermo Fisher Scientific (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Cellstar, by Greiner (1 mentions)
6 protocols using cellstar polypropylene tube
1
Sample Preparation for Elemental Analysis
2
Size-Exclusion Chromatography of Plasma Insulin
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Five hundred microlitres of plasma was loaded onto a HiLoad 16/60 Superdex 75 (120 ml) size‐exclusion column in combination with a 25 mmol/l Tris/0·52 mol/l NaCl buffer mobile phase at pH 7·4, with a flow rate of 1 ml/min. Optimization studies demonstrated that the addition of bovine serum albumin (BSA) to fraction collector tubes before GFC improved insulin recovery from the column, achieving >70%. Six millilitre elution volume fractions with 1 ml bovine serum albumin (BSA) (final volume 7 ml, calculated BSA concentration 40 g/l) were collected in Cellstar® polypropylene tubes (Greiner Bio‐One, Stonehouse, Gloucestershire, UK). A total of 36–114 ml eluted volume was collected.
GFC was performed using the ÄKTAprime plus™ liquid chromatography system (GE Healthcare, Uppsala, Sweden), and ultraviolet (UV) absorbance at 280 nm was recorded. The chromatography method demonstrated good precision with elution volume coefficient of variation of 6% for immunoglobulin (n = 30; mean elution volume 49 ml).
Samples were analysed for insulin using the LIAISON® XL immunoassay. This assay was chosen because in‐house data supported high analytical sensitivity (1·2 pmol/l) and acceptable coefficient of variation at lower insulin concentrations (8·6% at 34 pmol/l; n = 244).
GFC was performed using the ÄKTAprime plus™ liquid chromatography system (GE Healthcare, Uppsala, Sweden), and ultraviolet (UV) absorbance at 280 nm was recorded. The chromatography method demonstrated good precision with elution volume coefficient of variation of 6% for immunoglobulin (n = 30; mean elution volume 49 ml).
Samples were analysed for insulin using the LIAISON® XL immunoassay. This assay was chosen because in‐house data supported high analytical sensitivity (1·2 pmol/l) and acceptable coefficient of variation at lower insulin concentrations (8·6% at 34 pmol/l; n = 244).
3
Bacterial Reverse Mutation Ames Test Protocol
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The bacterial reverse mutation test (Ames test) was performed using the preincubation
method as described byMortelmans and Zeiger
(2000) , with slight alterations. Briefly, SalmonellaTyphimurium TA98 (Trinova, Gießen, Germany) was cultivated in nutrient broth (nutrient
broth no. 2; Oxoid, Wesel, Germany) overnight and then used at an OD of 0.9–1.0 in the
test. Bacteria were thereby incubated with top agar (Kobe I agar; Carl Roth), S9
originating from rat liver (Trinova) and sodium phosphate buffer (2.8 g/l
NaH2PO4·H2O, 12.8 g/l
Na2HPO4·2H2O, 2.7 g/l KCl, 1.8 g/l MgCl, 3.5 g/l
NADP, 1.6 g/l glucose-6-phosphate, pH = 7.4) for 20 min at 37 °C in 15 ml reaction
tubes (CELLSTAR polypropylene tubes; Greiner Bio-One; Frickenhausen, Germany). This
mixture was then poured into petri dishes (Sarstedt, Nümbrecht, Germany) containing
base agar (Kobe I agar, Carl Roth). Finally, the plates were incubated for 48 h at
37 °C and the colonies on each plate were counted. To test the stability of MelQx-M1
under the assay conditions, buffer containing 10% of rat liver S9 and 1 µM MelQx-M1
were incubated for 30 min at 37 °C, before the mixture was centrifuged and the
supernatant analyzed in the same manner as the samples described in
LC-ESI-MS2 section.
method as described by
(2000)
broth no. 2; Oxoid, Wesel, Germany) overnight and then used at an OD of 0.9–1.0 in the
test. Bacteria were thereby incubated with top agar (Kobe I agar; Carl Roth), S9
originating from rat liver (Trinova) and sodium phosphate buffer (2.8 g/l
NaH2PO4·H2O, 12.8 g/l
Na2HPO4·2H2O, 2.7 g/l KCl, 1.8 g/l MgCl, 3.5 g/l
NADP, 1.6 g/l glucose-6-phosphate, pH = 7.4) for 20 min at 37 °C in 15 ml reaction
tubes (CELLSTAR polypropylene tubes; Greiner Bio-One; Frickenhausen, Germany). This
mixture was then poured into petri dishes (Sarstedt, Nümbrecht, Germany) containing
base agar (Kobe I agar, Carl Roth). Finally, the plates were incubated for 48 h at
37 °C and the colonies on each plate were counted. To test the stability of MelQx-M1
under the assay conditions, buffer containing 10% of rat liver S9 and 1 µM MelQx-M1
were incubated for 30 min at 37 °C, before the mixture was centrifuged and the
supernatant analyzed in the same manner as the samples described in
LC-ESI-MS2 section.
4
Trace Element Sample Preparation
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Purification system (Milli-Q, Merck Millipore, Darmstadt, Germany) was used to provide purified water (18.2 MΩ cm). Nitric acid (HNO 3 ) Rotipuran p. a. ≥ 65% (Carl Roth, Karlsruhe, Germany) was subboiled with a MLS duoPUR (MLS, Leutkirch, Germany) prior to its use for the preparation of samples. For internal standards and preparation of calibration standards, we used ICP Single-Element Standards Certipur (Merck Millipore, Darmstadt, Germany) and Single Element Standards for ICP (Carl Roth, Karlsruhe, Germany). Fifteen and fifty mL Cellstar polypropylene tubes (Greiner Bio-One International GmbH, Kremsmünster, Austria) were used for preparation of all solutions.
5
Dental Plaque and Saliva Pooling
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Twelve patients were recruited from the Oral Health Unit, Green Lane Hospital, Auckland, New Zealand to participate in this study. All patients consented to providing sub- and supra-gingival dental plaque for this study, collected during a whole mouth scale. Plaque was removed by a registered dentist using sterile periodontal scalers and placed in a screw cap tube containing 1 mL of RNAlater® (AMBION, Inc., Austin, TX, USA). Samples were stored at -20°C until further processing. Twelve healthy volunteers from the University of Auckland, New Zealand consented to providing a fresh saliva sample. At least 1 mL of saliva was collected from each participant via passive secretion into a sterile container and frozen neat at -20°C. To create a homogenous sample of adequate volume for the comparison of multiple DNA extraction methods, 1 mL of each of the 12 plaque samples in RNAlater® (AMBION, Inc., Austin, TX, USA) was pooled in a 50 mL CELLSTAR® Polypropylene Tube (Greiner Bio-One) and vortexed until a consistent solution was achieved. In a similar manner, 1 mL of each of the 12 saliva samples was pooled and mixed thoroughly. The plaque and saliva pooled homogenates (totalling approximately 12 mL each) were divided separately into 200 μL aliquots for subsequent testing.
6
Cryopreserved HUVEC Cell Culture Protocol
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Commercially available, cryopreserved human umbilical vein endothelial cells (HUVEC), from pooled donors were acquired from Lonza (C2519A) and cultured in endothelial cell growth medium (EGM-2 BulletKit, CC-3162, Lonza) with 1% (v/v) gentamicin (10 ng mL -1 , Gibco). HUVECs at passage three were thawed and seeded in 175 cm 2 filter cap cell culture flasks (CELLSTAR, Greiner Bio-One). After 24 h, the cell culture media was exchanged and the HUVECs were expanded for the following 3 days before cell loading into the disc.
For generating a cell suspension for subsequent cell loading, adherent cells were washed with PBS (Dulbecco's phosphate buffered saline w/o calcium w/o magnesium, Biowest), detached by a 3 min incubation step at 37 °C using 0.05% (v/v) trypsin (Trypsin-EDTA Solution 10×, SIGMA Life Science) in Versene solution (Versene 1 : 5000 1×, Gibco). The cell suspension was transferred into a centrifuge tube (50 mL CELLSTAR polypropylene tube, Greiner Bio-One), trypsin inactivated by adding 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific) and centrifuged for 5 min at 1000 rpm or 216g (Multifuge 3S-R, Heraeus). Cells were counted using trypan blue (trypan blue 4 g l -1 in aqueous solution, VWR chemicals) using a hemocytometer (C-Chip Neubauer improved DHC-N01, NanoEnTek).
For generating a cell suspension for subsequent cell loading, adherent cells were washed with PBS (Dulbecco's phosphate buffered saline w/o calcium w/o magnesium, Biowest), detached by a 3 min incubation step at 37 °C using 0.05% (v/v) trypsin (Trypsin-EDTA Solution 10×, SIGMA Life Science) in Versene solution (Versene 1 : 5000 1×, Gibco). The cell suspension was transferred into a centrifuge tube (50 mL CELLSTAR polypropylene tube, Greiner Bio-One), trypsin inactivated by adding 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific) and centrifuged for 5 min at 1000 rpm or 216g (Multifuge 3S-R, Heraeus). Cells were counted using trypan blue (trypan blue 4 g l -1 in aqueous solution, VWR chemicals) using a hemocytometer (C-Chip Neubauer improved DHC-N01, NanoEnTek).
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