Cellular ROS levels were measured by flow cytometry using the florescent indicator 2',7'dichlorodihydrofluorescein diacetate (H2DCFDA; Thermo Fisher Scientific). After treatment, NGFDPC12 cells were incubated with 10 μM H2DCFDA in F‐12K medium for 25 min at 37°C. Cells were then detached by HyClone HyQtase (GE Healthcare), rinsed and analyzed with a Becton‐Dickinson FACSCalibur (Almaguel et al., 2009 ; Padilla et al., 2011 ). NGFDPC12 cells treated with 10 μM DL‐Buthionine‐[S,R]‐sulfoximine (BSO) for 24 hr served as positive control.
Hyclone hyqtase
Manufactured by GE Healthcare
Sourced in United States
HyClone HyQTase is a cell detachment reagent used in cell culture applications. It is designed to gently and effectively detach adherent cells from cell culture surfaces.
Automatically generated - may contain errors
Lab products found in correlation
Facsverse, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Lonza (2 mentions)
Caco 2, by Merck Group (2 mentions)
Ht 29, by Leibniz Institute DSMZ (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Mccoy 5a, by Lonza (2 mentions)
Hepes, by Lonza (2 mentions)
L glutamine, by Lonza (2 mentions)
Ht 29, by Lonza (2 mentions)
Facscalibur, by BD (1 mentions)
H2dcfda, by Thermo Fisher Scientific (1 mentions)
Hfls ra, by Cell Applications (1 mentions)
Synoviocyte growth medium, by Cell Applications (1 mentions)
Influx cell sorter, by BD (1 mentions)
Formaldehyde, by Polysciences (1 mentions)
Penicillin and streptomycin, by Lonza (1 mentions)
Caco 2, by Leibniz Institute DSMZ (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
β estradiol, by Merck Group (1 mentions)
9 protocols using hyclone hyqtase
1
Measuring Cellular Reactive Oxygen Species
2
Culturing Human Fibroblast-Like Synoviocytes
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HFLS-RA were purchased from Cell Applications, INC., (San Diego, CA, USA), and cultured in Synoviocyte Growth Medium (Cell Applications, CA, USA) according to the general instructions for culturing. The cells were incubated in a humidified chamber with 5% CO2 at 37 °C with the medium exchanged every 3 days. When the cells reached 80–90% confluence, they were detached with HyClone® HyQtase (GE, UT, USA) and replated at a ratio of 1:4. All the experiments used 3rd to 7th passages cells.
3
CD29-Based Cell Sorting in 3D Collagen
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Wild type MDA-MB-231 cells were grown in collagen I-coated tissue culture dished until 80% confluence. Cells were harvested using HyClone HyQtase (GE Healthcare Life Sciences, Marlborough, MA) and resuspended in FACS buffer (1% BSA, 0.5 mM EDTA in PBS). The cell suspension was then labeled using a monoclonal antibody against human CD29 (b1 integrin) conjugated to AlexaFluor 488. A cell suspension without added antibody was used as negative control. After labeling, the cells were analyzed within 1 h of detachment at the stem cell core of Sanford Consortium of Regenerative Medicine (La Jolla, CA) using a BD Influx cell sorter (BD, Franklin lakes, NJ). Cells were sorted based on fluorescence intensity into the top-expressing population (~15%, ITGB1 high) and bottom-expressing population (~13%, ITGB1 low). Sorted cells were replated into collagen-coated dishes and left to recover overnight. After recovery, the cells were embedded in 3D collagen gels as described above.
4
Endothelial Cell Surface Protein Expression
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HUVECs were grown to confluence in 12-well plates and treated with PT and TNFα as described. Afterwards, HUVECs were washed twice with pre-warmed PBS and detached with HyClone HyQTase (GE Healthcare). In the case of ICAM-1, detached HUVECs were fixed with 4 % formaldehyde (Polysciences, Hirschberg an der Bergstraße, Germany) in PBS (final concentration: 2 %) and incubated with FITC-labeled anti-human CD54 (ICAM-1) antibody (MCA1615F, Biozol, Eching, Germany) in PBS. In the case of VCAM-1, detached HUVECs were left unfixed and incubated on ice with PE-labeled anti-human CD106 (VCAM-1) antibody (555647, Becton Dickinson, Heidelberg, Germany) in PBS containing 0.2 % BSA. Protein expression on the endothelial surface was analyzed by flow cytometry (FACSVerse, Becton Dickinson).
5
Cultivation of Caco-2 and HT-29 Cells
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The human colonic epithelial cell lines Caco-2 (ACC 169) and HT-29 (ACC 299) were acquired from German Collection of Microorganisms and Cell Cultures (DSMZ) and grown at 37 °C in an incubator under oxic atmosphere with 5% CO2. Cells were passaged after reaching 80% confluence using HyClone HyQTase (GE Healthcare Life Sciences, Marlborough, MA, USA) to detach the cells and passage numbers up to 28 were used in the experiments. Caco-2 cells were cultivated in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with heat-inactivated (30 min at 56 °C) fetal bovine serum (20%; FBS; Gibco, UK), nonessential amino acids (1%, NEAA; Lonza, Bornem, Belgium), 15 mM HEPES (Lonza, Belgium), 100 U mL−1 penicillin and streptomycin (PEST; Lonza, Belgium) and 2 mM L-glutamine (Lonza, Belgium). HT-29 cells were grown in McCoy 5A (Lonza, Belgium) medium containing 10% FBS and 100 U mL−1 PEST.
6
Transdifferentiation and Mtb Infection of BLaER1 Cells
7
Measuring Cell Adhesion Molecule Expression
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HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase (GE Healthcare, Freiburg, Germany). In the case of ICAM-1 the detached cells were fixed with 4% formaldehyde (Polysciences, Hirschberg an der Bergstraße, Germany) in PBS for 10 min and washed once with PBS before incubating with the fluorescein isothiocyanate (FITC)-labeled anti-human CD54 (mouse, ICAM-1) antibody (MCA1615F, Biozol, Eching, Germany) at room temperature for 45 min. For all other proteins, the cells were not fixed and washed once with PBS before incubating with the antibodies phycoerythrin (PE)-labeled anti-human CD106 (mouse, VCAM-1), PE-labeled anti-human CD62E (mouse, E-selectin) and PE-labeled anti-human CD325 (mouse, N-cadherin) from Becton Dickinson on ice for 45 min. These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry (FACSVerse, Becton Dickinson, Heidelberg, Germany).
8
Culturing Caco-2 and HT-29 Intestinal Cells
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The human colonic epithelial cell lines Caco-2 (ACC 169) and HT-29 (ACC 299) were obtained from DSMZ and grown at 37°C in an incubator under oxic atmosphere with 5% CO2. Cells were passaged after reaching 80% confluence using HyClone HyQTase (GE Healthcare Life Sciences, USA) to detach the cells. Passage numbers 5–28 were used in the experiments. Caco-2 cells were grown in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with heat-inactivated (30 min at 56°C) fetal bovine serum (FBS, 20%; Integro B.V., Netherlands), non-essential amino acids (1%; Lonza, Belgium), 15 mM HEPES (Lonza, Belgium), 100 U ml-1 penicillin and streptomycin (PEST; Lonza, Belgium), and 2 mM L -glutamine (Lonza, Belgium). HT-29 cells were cultivated in McCoy 5A (Lonza, Belgium) medium containing 10% FBS and 100 U ml-1 PEST. For adhesion and ELISA assays, 10,000 Caco-2 or HT-29 cells per well were seeded onto 96-well microplates.
9
Expansion of Human Umbilical Cord Mesenchymal Stem Cells
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hUCMSCs were obtained from Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. hUCMSCs were maintained in low serum defined medium, which consisted of 56% low-glucose Dulbecco’s Modified Eagle Medium (DMEM-LG; Invitrogen, CA, USA), 37% MCBD 201 (Sigma, MO, USA), 2% fetal bovine serum (Thermo, Logan, UT), 0.5 mg/mL of AlbuMAX® I (Life technology, NY, USA), 1X Insulin-Transferrin-Selenium-A (Life technology, NY, USA), 10 nM Dexamethasone (Sigma, MO, USA), 10 ng/mL Epidermal growth factor (PeproTech, NJ, USA), 50 nM L-ascorbic acid 2-phosphate (Sigma, MO, USA), and 1 ng/mL of platelet-derived growth factor-BB (PeproTech, NJ, USA). The cells were incubated in a humidified chamber with 5% CO2 at 37 °C. Detaching with HyClone® HyQtase (GE, UT, USA) and replated at a ratio 1:4 when cells reached 70–80% confluence.
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