The freshly isolated Tie2+ cells after sorting were treated with various Growth factors and oxygen concentrations to test for culture conditions that could amplify and maintain the Tie2+ cells. Growth factors (Peprotech), including growth differentiation factor 5 (GDF5), GDF6, EGF, VEGF, FGF2 (100 ng/ml), and TGFβ1 (10 ng/ml), or coculture with IVD tissue using culture inserts (Becton Dickinson) for 6-well plates were applied to Tie2+ cells after sorting for 7 days in normoxia. The concentrations of the Growth factors were selected according to previously published results showing a beneficial effect on NPC and/or maintenance and proliferation of stem cells in vitro [19 (link)–25 (link)]. Hypoxic conditions at 2 % O2 have been shown in multiple studies [26 (link), 27 (link)], including by our group [19 (link), 28 (link)], to have a stimulatory effect on aggrecan expression by NPC. To test for cell proliferation and the conservation of Tie2 markers under hypoxia, Tie2– and Tie2+ cells were cultured in normoxia (atmospheric O2, ~21 %) or in hypoxia using a C-274-2 shelf chamber inside a standard incubator and 1× Pro-Ox controller (Biospherix, Union Street Parish, New York, USA) adjusted to 2 % O2 by addition of N2.
Culture insert
Manufactured by BD
Sourced in United States, France
The Culture Insert is a disposable, sterile laboratory product designed to facilitate cell culture and tissue engineering applications. It provides a three-dimensional matrix to support the growth and organization of cells in a controlled microenvironment.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (5 mentions)
Culture insert, by Corning (2 mentions)
Growth factor, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Shh n, by R&D Systems (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Glucose, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cmrl 1066 medium, by Corning (1 mentions)
Modfit lt, by BD (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Merck Group (1 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (1 mentions)
Cellquest, by BD (1 mentions)
6 well plate, by BD (1 mentions)
Panc 1, by BD (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Panc 1, by RIKEN BioResource Center (1 mentions)
16 protocols using culture insert
1
Tie2+ Cell Expansion and Maintenance
2
bLF Modulates Cellular Invasion
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HSC2 and HOC313 cells (6 × 105) were cultured in 6 cm dishes and incubated for 24 h. The medium was changed prior to pre-treatment of the cells with or without bLF for 72 h. Culture inserts of 8 μm pore size (Becton Dickinson Labware, NJ, USA) were inserted into 24-well plates, which contained 0.5 mL of medium. The inserts were then coated with 100 μL of Matrigel (1 mg/mL; BD Bioscience, Bedford, MA, USA). HOC313 cells (2 × 104) pre-treated with 100 μL of medium, in the presence or absence of bLF (1, 10, or 100 μg/mL), were added into the upper chambers and incubated at 37 °C. Stimulation with tPA (20 nM) and bLF (100 μg/mL) was performed in the same manner as for the wound-healing assay. The invasive cells were then fixed with 10% paraformaldehyde and stained with Mayer’s hematoxylin. The cells on the upper surface of the chambers were removed using a cotton swab. Finally, the membranes were separated from the insert and mounted on glass slides. The number of invading cells was counted under a microscope (200× magnification).
3
Biolactoferrin Modulates Cell Migration
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After trypsinization, 2.5 × 104 cells of 72 h pre-treated HSC2 and HOC313 cells with or without bLF (1, 10, or 100 μg/mL), were suspended in 200 µL of medium and placed into the upper chamber of culture inserts of 8 μm pore size (Becton Dickinson Labware, Franklin Lakes, NJ, USA). The inserts were then placed in a 24-well cell culture plate with bLF (0, 1, 10, or 100 μg/mL). The cultures of HSC2 cells were incubated for 6 h, and HOC313 for 12 h, at 37 °C. After the incubation, cells that had migrated to the lower side of the membrane were fixed with 10% paraformaldehyde and stained with Mayer’s hematoxylin. The cells on the upper surface of the filter were wiped off with a cotton swab. The migration cells that attached to the bottom of the membrane were counted under a microscope at 200× magnification.
4
Transwell Assay for Cell Migration
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Transwell methods and culture inserts (8 μM pore size, BD Biosciences) were used to analyze the cell migration. Cells (5 × 104/well) were plated into the upper chamber in serum-free medium, and the bottom chamber containing complete medium. After culturing for 24 h, cells were stained with 0.1% crystal violet, and the positive cells from five fields were counted and analyzed under a microscope.
5
Invasion Assay with Transwell Chambers
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Invasion ability was investigated by using Transwell invasion chambers. The culture inserts (8-μm pore size; BD Biosciences, Franklin lakes, NJ, USA) was precoated with 30 μg/insert Matrigel (BD Biosciences, Franklin lakes, NJ, USA) on top of the membrane. A total of 2.5 × 104 cells were seeded onto the upper chamber in medium containing 10% NuSerum. The lower chamber contained complete medium (RPMI supplemented with 10% FBS). The cells were allowed to invade toward the lower chamber overnight. On the second day, the invasive cells were fixed with methanol, stained with 50 μg/mL propidium iodide (Sigma, St. Louis, MO, USA) in ddH2O and analyzed using immunofluorescent microscopy. The amounts of invasive cells were quantified with ImageJ software.
6
Cerebellar Slice Culture Assay for Cellular Proliferation
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Postnatal cerebellar slice cultures were prepared by cutting 300 µm parasagittal sections using a
McIlwain tissue chopper, whereas entire coronal E16 cerebella primordia were used for embryonic explants. 8 to 10 slices or embryonic explants were plated on culture inserts (BD Becton Dickinson, Franklin Lakes, NJ, USA) and placed in a 6-well plates containing 1 ml serum-free slice culture medium. Cultures were kept at 37 °C in 5% CO 2 . Culture medium consists of Eagle's basal medium with Earles's salts (Life Technologies, Carlsbad, CA, USA) supplemented with glutamine (2 mM, Gibco, Carlsbad, CA, USA), glucose (32 mM, Sigma-Aldrich, St. Louis, MO, USA), penicillinstreptomycin (20 U/ml, Gibco), bovine serum albumin (10 mg/ml; Sigma, Saint Louis, MO, USA), and B27 supplement (Gibco). Cultures were treated with SHH-N (3 µg/ml, R&D Systems, Minneapolis, MN, USA) w/o cyclopamine (Tocris Bioscences, Bristol, UK), and fixed after 24 hours (1 day in vitro -DIV-) or 48 hours (2 DIV). To label proliferating cells, bromodeoxyuridine (BrdU, 10 µM, Sigma Aldrich) was applied at 22 hours and washed out after 2 hours. Slices were fixed immediately thereafter or at 2 DIV (Fig. 1 a). Untreated slices incubated with BrdU were used as control.
McIlwain tissue chopper, whereas entire coronal E16 cerebella primordia were used for embryonic explants. 8 to 10 slices or embryonic explants were plated on culture inserts (BD Becton Dickinson, Franklin Lakes, NJ, USA) and placed in a 6-well plates containing 1 ml serum-free slice culture medium. Cultures were kept at 37 °C in 5% CO 2 . Culture medium consists of Eagle's basal medium with Earles's salts (Life Technologies, Carlsbad, CA, USA) supplemented with glutamine (2 mM, Gibco, Carlsbad, CA, USA), glucose (32 mM, Sigma-Aldrich, St. Louis, MO, USA), penicillinstreptomycin (20 U/ml, Gibco), bovine serum albumin (10 mg/ml; Sigma, Saint Louis, MO, USA), and B27 supplement (Gibco). Cultures were treated with SHH-N (3 µg/ml, R&D Systems, Minneapolis, MN, USA) w/o cyclopamine (Tocris Bioscences, Bristol, UK), and fixed after 24 hours (1 day in vitro -DIV-) or 48 hours (2 DIV). To label proliferating cells, bromodeoxyuridine (BrdU, 10 µM, Sigma Aldrich) was applied at 22 hours and washed out after 2 hours. Slices were fixed immediately thereafter or at 2 DIV (Fig. 1 a). Untreated slices incubated with BrdU were used as control.
7
Induction of Pancreatic Cancer EMT
8
Co-culture of Human PSCs and PDACs
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Human PSC cells (1–10×105 cells/well) were seeded in 24 well or 6 well culture plates (BD Bioscience) in DMEM medium supplemented with 10% FBS, 1% penicillin/streptomycin. 1:1 proportion of PDAC cells (1–10×105 cells/culture insert) were seeded into the culture inserts of 3 μm pore size (BD Bioscience) in appropriate media supplemented with 10% FBS, 1% penicillin/streptomycin. The culture inserts seeded with PDACs were placed into the 24/6-well plates containing PSC cells, and incubation was conducted for 2 days under standard culture conditions (5% CO2, at 37°C).
9
Schwann Cell Migration Assay
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Schwann cell migration was studied using the sNF96.2 cell line under various conditioned media on Boyden chambers. Culture inserts (BD, Le pont le Claix, France) with a porous membrane at the bottom (8 μ pores) were coated with a mix made of gelatin 0.1% and fibronectin 10 μg/ml, and then were seeded with sNF96.2 (100 000 per insert) and placed into wells containing the conditioned media. Migration was performed for 4 h. After cleaning and briefly staining inserts with coomassie blue, migration was assessed by counting the number of colored cells in 10 high-power fields (magnification x20).
10
Cell Proliferation, Apoptosis, and Transwell Assays
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The cell proliferation activity was assessed by EdU (5-ethynyl-2′-deoxyuridine) incorporation and flow cytometry. The EdU assay was performed according to the manufacturer′s instructions (RiboBio, Guangzhou, China), and the percentage of EdU-positive cells was used to evaluate cell proliferation. For flow cytometry, the cells were stained with 2 µM CFSE (Sigma, USA). Data were analyzed using Cell Quest and ModFitLT software (BD, USA), and the proliferation index was calculated as previously described31 (link). For apoptosis, the cells were assayed using a FITC AnnexinV Apoptosis Detection Kit (BD, USA) followed by flow cytometry analysis. For in vitro transwell assays, 1 × 105 cells were seeded in the upper chamber of a culture insert (8-mm pore size in 24-well plates, BD, USA) without (migration assay) or with (invasion assay) Matrigel (BD, USA) in serum-free media. The bottom chamber was filled with the same medium supplemented with 10% fetal bovine serum. After 24 h, the cells on the undersurface of the membrane were stained with crystal violet and counted using a light microscope. The cell migration and invasion properties were evaluated by the number of cells per high power field. All experiments were performed in triplicate and with at least three biological replicates.
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