Parental RM1 cells were seeded at 1–2.5 × 104 cells per well in a 24‐well plate. BM was derived as previously specified, and ~ 8 h later, 6 × 105 cells were added (±) to cultures for 48 h ± MS275 (1 μM). Excess naïve BM was stored at −80°C for RNA extraction to assess IRG expression. For transwell cultures, BM was ± seeded onto 0.4‐μm filters (Corning) in a 24‐well plate and cultured for 48 h. For CD11b+ co‐cultures, CD11b+ Ly6G+ populations were FACS‐isolated from naïve BM after staining as described. At all co‐culture endpoints, RM1 cells were pelleted for RNA extraction and qRT–PCR by methods previously outlined.
0.4 μm filter
Manufactured by Corning
Sourced in United States
The 0.4 μm filter is a laboratory filtration device designed to remove particles and contaminants from liquids or gases. It features a pore size of 0.4 micrometers, which allows the passage of smaller molecules while effectively trapping larger particulates. This filter can be used for a variety of applications that require high-precision filtration.
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5 protocols using 0.4 μm filter
1
RM1 Cell Co-culture with Bone Marrow
2
Evaluating NHLF Effects on Mesothelioma
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To assess the effects of NHLF on mesothelioma cell proliferation, NHLF (1×104 each chamber) were seeded into the upper chamber (0.4 μm filters, Corning) and mesothelioma cells (1×104 each chamber) were plated in the lower chamber of transwell systems. After incubation of 3, 5 or 7 days, cells from each chamber were collected and the cell suspension was counted with a standard hemocytometer. Using the same method, mesothelioma cells (1×105 per chamber) and NHLF (1×105 per chamber) were seeded and separately collected after 48 h-incubation and used to prepare lysates for western blot analysis. Relative CTGF expression in comparison to each mono-culture experiment was calculated after semiquantitative analysis with ImageJ.
We performed migration and invasion assays in the same way mentioned above. Mesothelioma cells (1×104 per chamber) were seeded into the upper chamber, and NHLF (1×104 per chamber) were plated in the lower chamber of transwell systems. To assess the effect of NHLF, mesothelioma cells were cultured with or without NHLF. To analyze the effect of drugs on the migration/invasion behavior of mesothelioma cells in circumstances similar to in vivo, migration/invasion assays were performed in co-culture conditions.
We performed migration and invasion assays in the same way mentioned above. Mesothelioma cells (1×104 per chamber) were seeded into the upper chamber, and NHLF (1×104 per chamber) were plated in the lower chamber of transwell systems. To assess the effect of NHLF, mesothelioma cells were cultured with or without NHLF. To analyze the effect of drugs on the migration/invasion behavior of mesothelioma cells in circumstances similar to in vivo, migration/invasion assays were performed in co-culture conditions.
3
Differentiation of EPC2-hTERT Airway Epithelial Cells
4
HUVEC culture with SWCNT aggregates
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HUVECs were used throughout this study and cultured as described previously.45 (link) HUVECs, endothelial basal medium (EBM), and supplements (EGM-MV: 5% fetal bovine serum, 12 μg/mL bovine brain extract, 1 μg/mL hydrocortisone, and 1 μg/mL GA-1000) were purchased from Lonza (USA). Cells were grown on 30 μg/mL bovine collagen type-I (BD Biosciences)-coated plates in a humidified incubator with 5% CO2 at 37 °C. A hypoxic chamber (Billups-Rothenberg Inc.) was filled with 2% O2 and placed within the tissue culture incubator. HUVECs were grown to passage 4 at ∼80% confluence and used for all experiments. Complete media consisted of EBM, 5% FBS, and all EGM-MV supplements, while starving media had reduced FBS levels of 0.5%. Serum-free media contained no additives. Conditioned media was made by incubating ∼80% confluent cells with starving media for 10 h. The resulting supernatants were filtered with a 0.4 μm filter (Corning, USA) to remove debris. Unless otherwise stated, aggregates were formed via introduction of (AT)15–SWCNT in 0.1 M NaCl at 2 μg/mL to test solutions and incubated under culture conditions for 6 h.
5
Derivation and Maintenance of Mouse ESCs
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The mouse green fluorescent protein (GFP) expressing-ESC line (F12) derived from a C57BL/6 mouse was kindly donated by Professor Melitta Schachner (Rutgers University). As previously described, freshly thawed ESCs (P0) were seeded into a 100 mm tissue culture dish with mitomycin-treated murine embryo fibroblasts (MEFs) as feeder layer. ESC culture medium was composed of 103 U/ml Leukemia Inhibitory Factor (LIF) (Millipore, CA, USA), 15 % FBS, 1 % non-essential amino acids solution (MEM), 200 mM L-glutamine, 1 % nucleoside solution, 1 % 100nM Na-Pyruvate, 0.2 % 2-β-Mercaptoethanol and DMEM. After 3–4 passages, ESCs were transferred to a 0.1 % gelatin-coated tissue culture dish without a feeder layer, and sub-cultured every 2–3 days prior to collection of the conditioned ESC media (ESC-M). The collected ESC supernatant was spun at 2500 RPM for 10 min and then filtered through a 0.4-μm filter (Corning, USA) to remove any debris. ESC-M was collected in this manner from multiple passages and pooled and stored at −80 °C prior to use. Control culture medium (without ESCs) was used as control medium (CON-M) in this study.
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