3D aggregates were formed as described previously (27 (link), 28 (link)). Briefly, normal human lung fibroblasts (NHLF) and bronchial smooth muscle cells (BSMC) were isolated from anonymous donors of different ages and sexes and were purchased from Lonza (Basel, Switzerland). All cells were cultured at 37°C and 5% CO2 in primary cell culture media. NHLF, BSMC and LAM cell types were sub-cultured and mixed at 1:1 ratio then dispensed 3*105 cells/well onto a low-attachment 96-well U-bottom plates (Corning, New York, USA). The 3D aggregate co-cultures were incubated in the presence or absence of 10 nM rapamycin and/or 2 μM RA for 24 h, then collected into cryomold and sectioned for staining.
Low attachment 96 well u bottom plate
Manufactured by Corning
Sourced in United States
The Low Attachment 96-well U-bottom Plate is a laboratory product designed to create a non-adherent environment for cell culture applications. The U-shaped well bottom promotes cell aggregation and the low-attachment surface minimizes cell attachment, making it suitable for spheroid and suspension cell culture.
Automatically generated - may contain errors
Lab products found in correlation
Hmvec l, by Corning (1 mentions)
Recombinant human wnt5a, by R&D Systems (1 mentions)
Hmvec l, by Lonza (1 mentions)
Matrigel, by Corning (1 mentions)
Ultra low attachment 6 well plate, by Corning (1 mentions)
Stemdiff cerebral organoid kit, by STEMCELL (1 mentions)
Y 27632, by Merck Group (1 mentions)
6 protocols using low attachment 96 well u bottom plate
1
3D Lung Cell Aggregates Formation
2
3D Airway Epithelial-Fibroblast Aggregates
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Normal primary human small airway epithelial cells (SAEC) and normal human lung fibroblast (NHLF) were purchased from Lonza (Basel, Switzerland), isolated from anonymous donors of different ages and sexes. All cells were cultured at 37 °C and 5% CO2 in primary cell culture media. Both cell types were sub-cultured and mixed at 1:1 ratio then dispensed 3*105 cells/well onto a low-attachment 96-well U-bottom plates (Corning, New York, USA) (Additional file 1 : Figures S3 and S4). 3D aggregates were formed as described previously (Kovacs et al., 2014). Aggregates were treated with 0.1 μg/ml of recombinant human protein Wnt4, Wnt5a or Wnt7a, respectively for 48 h, then collected for total RNA isolation for TaqMan based PCR application (n = 3 biological repeats).
3
Lung Aggregate Tissue Generation
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Three-dimensional lung aggregate tissues were generated as described in previous studies (30 (link), 43 (link), 44 (link)). Briefly, NHLF, SAEC, and HMVEC-L all purchased from Lonza (Lonza, Basel, Switzerland) were mixed (40% NHLF, 30% SAEC, and 30% HMVEC-L) in the absence or presence of peripheral monocyte-derived macrophages (30% NHLF, 25% SAEC, 25% HMVEC-L, and 20% macrophages) as described, then the cell suspensions were dispensed onto a low attachment 96-well U-bottom plate (Corning, New York, USA) and were centrifuged at 600 g for 10 min at room temperature. Tissue aggregates were maintained in 2:2:1 ratio of endothelial cell growth medium, SAGM, and fibroblasts growth medium. In the various experiments, aggregates were treated with 0.5% CSE or 1 µg/ml recombinant human Wnt5a (R&D Systems, Minneapolis, USA) for 48 h.
4
Cerebral Organoid Generation from Human iPSCs
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Organoids were generated using a STEMdiff Cerebral Organoid Kit assay following the manufacturer's instruction. On day 0, human iPSCs at 90% confluence were dissociated into single cells using Accutase (5 minutes, 37°C). After centrifugation at 1000g for 5 minutes, iPSCs were resuspended in EB Formation Medium with 10 μM Rock Inhibitor Y27632 and diluted to the concentration of 9 × 105 cells per mL. Then, 100 μL of cell suspension was distributed into each well of a low‐attachment 96‐Well U‐bottom plate (Corning) to form single EBs, medium was changed every two days. On days 5‐6, half of the medium was replaced with induction medium. On day 7, organoids were harvested and embedded in Matrigel (Corning) and continued to grow in expansion medium in suspension culture in ultra‐low attachment 6‐well plates (Corning). After 3 days of maintenance, embedded organoids were cultured in maturation medium and the plates were transferred to a shaker for the continuous culturing, medium was changed every 3 days.
5
Mixed Cell Co-Culture Assay
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NHLF and A549 or PC9 were mixed in 1:1 ratio and a total of 30,000 cells/well were pipetted onto a low-attachment 96-well U-bottom plate (Corning, NY, USA). Cells were sedimented (600 g for 10 min) and cultured at 37 °C and 5% CO2 in mixed DMEM:FGM-2 or RPMI:FGM-2 media at 1:1 ratio, respectively [14 (link)].
6
Cerebral Organoid Generation from hiPSCs
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Organoids were generated using a STEMdiff Cerebral Organoid Kit (STEMCELL Technologies; 08570) assay following the manufacturer’s instructions. On day 0, hiPSCs at 90% confluence were dissociated into single cells using Accutase (5 min, 37 °C). The hiPSCs were resuspended in embryoid body formation medium with 10 μM Y27632 (Sigma-Aldrich Co., Seoul, Korea; Y503), a Rock inhibitor, and diluted to a concentration of 9 × 103 cells per mL after centrifugation at 1000× g for 5 min. Then, 100 μL of cell suspension was distributed into each well of a low-attachment, 96-well, U-bottom plate (Corning) to form single EBs. The medium was changed every two days.
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