A549 cells pretreated with W-BR, E-BR, SSa, and SSd for 24 h were harvested using TrypLE™ Express solution (Thermo Fisher Scientific) and resuspended in a serum-free medium at 2 ×105 cells/mL. Cells (2 × 104 cells/well) were added to the wells of collagen I, collagen IV, or poly-d-lysine/laminin-coated 96-well culture plates (Corning Life Sciences, Kennebunk, ME, United States). After incubation for 30 min, the cells were washed three times with phosphate-buffered saline to remove unbound floating cells, and the attached cells were stained with a crystal violet solution (0.2% crystal violet in 20% methanol) for 30 min at room temperature. After washing with distilled water, stained cells were dissolved in 200 μl of 1% sodium dodecyl sulfate solution, and the absorbance at 590 nm was measured using a SpectraMax3 microplate reader (Molecular Devices, LLC).
Tryple express solution
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany
TrypLE Express solution is a cell dissociation reagent designed to gently and effectively detach adherent cells from cell culture vessels. It is a recombinant trypsin-like enzyme that can be used as a substitute for traditional trypsin-EDTA solutions.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (5 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Bz x710 microscope, by Keyence (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Novocyte 2060r flow cytometer, by Agilent Technologies (2 mentions)
Growth factor reduced matrigel, by Corning (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Wi 26 va4, by American Type Culture Collection (2 mentions)
Advanced mem, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Spectramax3 microplate reader, by Molecular Devices (1 mentions)
Novoexpress software, by Agilent Technologies (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
57 protocols using tryple express solution
1
Quantifying Cell Adhesion on Coated Surfaces
2
FGF2 Apoptosis Induction in NIH3T3 Cells
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The apoptosis study was carried out by flow cytometry using NovoCyte 2060R Flow Cytometer (ACEA Biosciences, San Diego, CA, USA) and the Annexin V Apoptosis Detection Kit FITC (Thermo Fisher Scientific, Waltham, MA, USA). For the analysis, 100,000 NIH3T3 cells were seeded onto each well of a 12-well culture plate and allowed to adhere overnight. The cells were subsequently starved for 8 h with serum-free medium, and then, treated with 10 ng/mL FGF2 WT or dimers in the presence or absence of 10 U/mL heparin. After 72 h of incubation, the cells were harvested with TrypLE Express solution (Thermo Fisher Scientific), pelleted and rinsed extensively with PBS. Annexin V-FITC and propidium iodide staining was performed according to the manufacturer’s protocol. Excessive dyes were washed off with PBS and samples were subjected to FACS analysis. The data were analyzed with NovoExpress Software (ACEA Biosciences, San Diego, CA, USA).
3
Isolation and Expansion of Murine Liver Organoids
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Murine organoids were isolated from adult C57BL/6J mice, KraslslG12D mice, or KraslslG12D/wt; p53lox/lox mice according to published protocols.11 Briefly, murine livers were minced and enzymatically digested in Earle’s Balanced Salt Solution (EBSS; Thermo Fisher, Waltham, MA) containing 2.5 mg/mL Collagenase Type IV (Sigma‐Aldrich, St. Louis, MO) and 0.1 mg/mL DNase I (Sigma‐Aldrich) for 20 to 40 minutes with repeated pipetting and passed through a 70‐µm cell strainer. After additional washes, cells were spun at 300g, resuspended in 100% Growth Factor Reduced Matrigel (Corning, NY), and plated (two 50‐µL droplets per 24‐well). After solidification, Matrigel droplets were overlaid with 500‐µL murine liver organoid media according to published protocols.11 Alternatively, residual fragments retained in the cell strainer after collagenase digest were plated in Matrigel. For passaging, organoids were mechanically disrupted by repeated pipetting using a P200 pipette tip, followed by a 5‐minute to 8‐minute enzymatic digestion in TrypLE Express solution (Thermo Fisher).
4
Murine Gallbladder Organoid Isolation and Culture
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Murine gallbladder organoids were isolated from adult C57BL/6J mice or KraslslG12D mice with some modifications to published protocols [47 (link)]. Briefly, the murine gallbladder was minced with a scalpel and filtered through a 100 µm mesh. After additional washes with PBS, cells were spun at 300 g for 5 min, resuspended in 100% Growth Factor Reduced Matrigel (Corning, NY, USA), and plated in a 24-well plate (two 50 µL droplets per well). After solidification, Matrigel droplets were overlaid with 500 µL murine liver organoid media according to published protocols [47 (link)]. For passaging, organoids were mechanically disrupted by repeated pipetting using a P200 pipette tip, followed by a 3- to 5-minute enzymatic digestion in TrypLE Express solution (Thermo Fisher, Waltham, MA, USA).
5
HepG2 Cell Culture Protocol
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The HepG2 cell line was purchased from ATCC (HB-8065) and cultured in accordance to the manufacturer’s protocol. Cell culture medium consisted of Dulbecco’s Modified Eagle’s Medium (DMEM), low glucose w/l -glutamine, w/sodium pyruvate (Biowest, Riverside, MO, USA) supplemented with 10% Fetal Bovine Serum (FBS, Sigma Aldrich, Munich, Germany). Cells were cultured in aseptic and unchanging conditions in an incubator (37 °C in a humidified 5% CO2 atmosphere). Cells were passaged with TrypLE Express solution (ThermoFisher, Warsaw, Poland). The media were changed every second day.
6
Clonogenic Assay for Radiation and Chemotherapy
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Sub-confluent HMLE cells were trypsinized using TrypLE express solution (Thermo Fisher Scientific, Inc.). Live cells were counted using an automated cell counter (TC20; Bio-Rad Laboratories, Inc.) and trypan blue exclusion. In the IR group, cells were immediately irradiated in suspension to generate a dose curve of 0, 2, 4 and 6 Gy and colony-forming assay was performed immediately following IR by plating cells in 60-mm Petri dishes, in triplicate.
In the drug treatment group, following trypsinization and counting, cells were plated in 60-mm diameter Petri dishes, in triplicate. At 6 h after plating, drug treatment (4 to 16 µM exposition to 5FU, 2 to 6 µM exposition to Cisplatin and 1 to 3 nM exposition to Paclitaxel) was performed for three days, then cells were washed (PBS) and incubated for 7 days with fresh medium.
Number of cells seeded increased with radiation dose or drug concentration but was identical for each cell line tested. After 7 days, cells were fixed at room temperature for 30 min in 4% paraformaldehyde, washed (PBS) and stained at room temperature for at least 2 h in methylene blue/30% methanol. Colonies containing >50 cells were manually counted. The surviving fraction at each radiation dose was normalized to that of the non-irradiated sample and points were fitted using an exponential tendency curve. At least three independent experiments were performed.
In the drug treatment group, following trypsinization and counting, cells were plated in 60-mm diameter Petri dishes, in triplicate. At 6 h after plating, drug treatment (4 to 16 µM exposition to 5FU, 2 to 6 µM exposition to Cisplatin and 1 to 3 nM exposition to Paclitaxel) was performed for three days, then cells were washed (PBS) and incubated for 7 days with fresh medium.
Number of cells seeded increased with radiation dose or drug concentration but was identical for each cell line tested. After 7 days, cells were fixed at room temperature for 30 min in 4% paraformaldehyde, washed (PBS) and stained at room temperature for at least 2 h in methylene blue/30% methanol. Colonies containing >50 cells were manually counted. The surviving fraction at each radiation dose was normalized to that of the non-irradiated sample and points were fitted using an exponential tendency curve. At least three independent experiments were performed.
7
Isolation and Dispersion of Neonatal Porcine Islet-Like Cell Clusters
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NPICCs were isolated from pancreata (n = 12) of 2–5 days-old piglets by collagenase digestion as described previously (22 (link), 23 (link)). Cell clusters were cultured in RPMI 1640 (PAN-Biotech, Aidenbach, Germany), 2% human serum albumin (Takeda, Konstanz, Germany), 10 mM nicotinamide, 20 ng/ml exendin-4 (Merck, Darmstadt, Germany) and 1% antibiotic-antimycotic (Thermo Fisher Scientific, Germering, Germany) (basal islet culture [B-IC] medium). On day 4, NPICCs were harvested and islet equivalents (IEQ) were determined under a stereomicroscope. NPICCs were either re-cultivated in B-IC medium (control group) or washed with PBS and incubated in TrypLE Express solution (Thermo Fisher Scientific) for 8–12 min at 37°C with gentle mixing every 60 s until dispersion into single cells was observed under a stereomicroscope. Then, the cells were filtered through a 40-µm filter (Corning, Wiesbaden, Germany) to remove debris.
8
Singularization and Flow Cytometric Analysis of Spheroids
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For the flow cytometric analysis, spheroids were washed with PBS, transferred into 2 ml reaction tubes and 200 μl TrypLE Express solution (Thermo fisher, Germany) was added for enzymatic digestion. Cells were incubated at 37°C. Additionally, every 10 min cell separation was stimulated by mechanical disruption through rough pipetting using a glass pipette. After 20 min of incubation, another 200 μl TrypLE Express solution was added to the reaction tube. This process was performed until up to 1 h, or ended earlier, if cells were already singularized. The cells were centrifuged (5 min, 200xg) and the pellet resuspended in PBS for flow cytometric analysis. The flow cytometer BD FACSAria™ Fusion (Becton Dickinson, USA) was used to evaluate single cell fluorescence. The target cell population was excited at 488 nm and fluorescence detected via FL 1 detector (533/30 nm) to analyze the cellular expression of UnaG protein.
9
Apoptosis Analysis in Resistant Breast Cancer
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Analysis of
the mechanism of cell death was carried out as previously described.44 (link) Briefly, 100,000 MCF7-R1 cells were seeded into
each well of a 12-well culture plate, allowed to adhere overnight,
and treated with 10 nM MMAE or conjugates in the presence of 10 U/mL
heparin for 72 h. Then the cells were harvested with a TrypLE Express
solution (Thermo Fisher Scientific), stained by Annexin V-FITC and
propidium iodide (according to the manufacturer’s protocol),
and analyzed by flow cytometry using a NovoCyte 2060R flow cytometer
(ACEA Biosciences, San Diego, CA).
the mechanism of cell death was carried out as previously described.44 (link) Briefly, 100,000 MCF7-R1 cells were seeded into
each well of a 12-well culture plate, allowed to adhere overnight,
and treated with 10 nM MMAE or conjugates in the presence of 10 U/mL
heparin for 72 h. Then the cells were harvested with a TrypLE Express
solution (Thermo Fisher Scientific), stained by Annexin V-FITC and
propidium iodide (according to the manufacturer’s protocol),
and analyzed by flow cytometry using a NovoCyte 2060R flow cytometer
(ACEA Biosciences, San Diego, CA).
10
Transwell Assay to Measure Cell Migration
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