Cells were harvested from confluent flasks, using Hanks Balanced Salt Solution (Gibco/Life Technologies, Thermo Fisher Scientific) and Trypsin‐EDTA 1X (Gibco), and transferred to petri dishes with cover slips. Once the cells had reached 50%–60% confluency, the cover slips were harvested using standard methods (ACT Cytogenetics Laboratory Manual, 2nd edition, Barch, MJ, ed. Raven Press, 1991); briefly, exposure to colcemid for 30 mintes (Gibco), followed by warm hypotonic solution (0.7% sodium citrate) for 20 minutes, then by a 2‐minute pre‐fixation in 3:1 Carnoy's fixative (3:1 methanol: glacial acetic acid) and then three additional, 10‐minute washes with the same fixative. The metaphase spreads were then GTG‐banded using standard methods 11 . The cultured cells were analyzed following the College of American Pathologist clinical guidelines for non‐neoplastic disorders; namely, 20 metaphase cells were counted and a minimum of five cells were analyzed band for band and karyotyped.
Trypsin edta 1x
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Australia, Germany
Trypsin-EDTA (1X) is a cell dissociation reagent used to detach adherent cells from cell culture surfaces. It contains the proteolytic enzyme trypsin and the chelating agent EDTA, which disrupt cell-cell and cell-substrate adhesions, enabling the release of cells from the culture vessel.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (21 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (16 mentions)
Glutamax, by Thermo Fisher Scientific (6 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Penicillin streptomycin, by Merck Group (3 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Dmem high glucose, by Merck Group (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Hepes ph 7, by Corning (2 mentions)
10 cm tissue culture plate, by Genesee Scientific (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Aria 3, by BD (2 mentions)
Dmem f12, by Merck Group (2 mentions)
Hyaluronidase, by Merck Group (2 mentions)
Tryple express, by Thermo Fisher Scientific (2 mentions)
Aria iiu, by BD (2 mentions)
81 protocols using trypsin edta 1x
1
Cytogenetic Analysis of Cultured Cells
2
Dual Reporter Mouse Embryonic Stem Cells
3
Microsphere-Encapsulated Cell Culture Assay
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Every 200 microspheres were encapsulated with 2.5e5 cells with different NBC: MSC ratios at day 0, and they were cultured for 7, 14, and 21 days. At each time point, 200 microspheres from each group were digested enzymatically by collagenase from Clostridium histolyticum (Sigma) at 200 units/ml for 45–60 min. Single cells suspensions were obtained by treating the digested aggregates with 0.25% trypsin-EDTA(1X) (Gibco) before numeration by trypan blue assay. The growth of cells in microspheres could then be calculated.
4
Gastric Cancer Cell Lines Cultivation
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Four human gastric carcinoma cell lines (AGS SORE6−, AGS SORE6+, Kato III SORE6−, and Kato III SORE6+) established under the scope of previous work [7 (link)] were used in this study. Furthermore, the human gastric carcinoma cell lines AGS (ATCC, CRL-1739, Manassas, VA, USA) and Kato III (ATCC, HTB-103, Manassas, VA, USA), from which the SORE6 cell lines were derived, were also used, as well as the human embryonic kidney cell line, HEK293T (kindly provided by Dr. João Relvas from Glial Cell Biology Group, i3S, Porto, Portugal). All gastric cancer cell lines were cultured in RPMI medium 1640 with 25 mM HEPES and GlutaMAX-1 (Gibco, Life Technologies, Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Biowest, Nuaillé, France). The HEK293T cell line was cultured in DMEM with 4.5 g/L D-glucose, L-glutamine, and pyruvate (Gibco, Life Technologies, Thermo Fisher) supplemented with 10% FBS. All cells were maintained at 37 °C and 5% (v/v) CO2. When the cell cultures reached a confluence of around 80% they were trypsinized with 0.05% Trypsin-EDTA (1X) (Gibco, Life Technologies, Thermo Fisher) and sub-cultured or used in cell-based assays.
5
Clonogenic Assay for Cell Lines
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H520 and A549 were suspended with pancreatin (0.25% Trypsin-EDTA 1X; Gibco; Thermo Fisher Scientific, Inc.) and counted. The cells were then added into 60-mm dishes (1,000 cells/dish) containing 5 ml culture medium and incubated at 37˚C in an atmosphere containing 5% CO2 and saturated humidity for 7-14 days. Cell culture was terminated when macroscopic cell colonies were visible on the dishes. After discarding the medium, PBS was used to wash the cells, which were then fixed with 4% paraformaldehyde for 30 min at 25˚C and stained with 0.1% crystal violet for 30 mins at 25˚C. Finally, excess dye was gently washed from the cells with running water and the cells were observed under an optical microscope (magnification, x400; Leica Microsystems GmbH). Colony images were obtained by scanning the culture dishes.
6
Uptake of Amine-Functionalized TiO2 Nanoparticles by HaCaT Cells
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HaCaT cells were seeded in 24-well culture plates with a total of 1.5×104 cells per well and incubated at 37 °C and 5 % CO2. After 24 h incubation the cells were exposed to amine functionalised TiO2-NP concentrations of 0.5, 1 and 4 mM for 24 h. Subsequently, the culture medium was removed and the cells were rinsed with PBS and harvested using 0.05 % trypsin-EDTA (1X) (Gibco by Life Technologies Pty. Ltd., Mulgrave, VIC, Australia). The solution was centrifuged at 300 g for 5 min and the cells were resuspended in PBS (2 mL). The uptake of TiO2-NPs by HaCaT cells was determined by flow cytometry (FACS Canto II, BD Biosciences). 10,000 gated events were counted and forward and side-scatter were recorded. Changes in side-scatter were assessed relative to an untreated control cell population.
7
Isolation of Embryonic Germ Cell Progenitors
8
Culturing H9c2 Rat Cardiomyoblasts
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The H9c2 rat cardiomyoblast cell line was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) (Cat# CRL-1446) and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). Cells were then incubated at 37 °C in humidified atmosphere with 5% CO2. After reaching ~80% of density in 100-mm dishes, cells digestion was performed using 0.25% Trypsin-EDTA (1X) (Gibco) according to a 1:2 ratio, following manufacturer’s instructions (ATCC). To perform experiments, cells were seeded and incubated for 48 h at 37 °C, 95% O2 and 5% CO2, as previously reported [34 (link),35 (link),36 (link),37 (link)].
9
Isolation of Embryonic Germ Cell Progenitors
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PGC isolation was carried out as previously described4 (link). Briefly, the embryonic trunk (E10.5) or genital ridge (E11.5-E14.5) was digested at 37°C for 3 min using 0.05% Trypsin-EDTA (1x) (Gibco) or TrypLE Express (Thermo). Enzymatic digestion was followed by neutralization with DMEM/F-12 (Gibco) containing 15% foetal bovine serum (Gibco) and manual dissociation by pipetting. Following centrifugation, cells were re-suspended in DMEM/F-12 supplemented with hyaluronidase (300 µg/ml; Sigma), and a single cell suspension was generated by manual pipetting. Following centrifugation, cells were re-suspended in ice-cold PBS supplemented with poly-vinyl alcohol (10 µg/ml) and EGTA (0.4 mg/ml, Sigma). GFP positive cells were isolated using an Aria IIu (BD Bioscience) or Aria III (BD Bioscience) flow cytometer and sorted into ice cold PBS supplemented with poly-vinyl alcohol (10 µg/ml) and EGTA (0.4 mg/ml, Sigma).
10
Generation of Neuroblastoma Cell Lines
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The wild type human SK-N-SH neuroblastoma cell line was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and used for the generation of our screening cell lines.
The wild type mouse Neuro 2A (N2a) was purchased from American Type Culture Collection (ATCC). All cell lines were cultivated in DMEM Glutamax (Gibco) supplemented with 1% penicillin/streptomycin (Gibco) and 10% inactivated FBS (Sigma-Aldrich), respectively. For detachment, cells were treated with 0.05% Trypsin–EDTA 1x (Gibco) for 10 min at 37 °C. All compounds were purchased from Selleckchem.
The wild type mouse Neuro 2A (N2a) was purchased from American Type Culture Collection (ATCC). All cell lines were cultivated in DMEM Glutamax (Gibco) supplemented with 1% penicillin/streptomycin (Gibco) and 10% inactivated FBS (Sigma-Aldrich), respectively. For detachment, cells were treated with 0.05% Trypsin–EDTA 1x (Gibco) for 10 min at 37 °C. All compounds were purchased from Selleckchem.
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