A predefined number of cells was seeded in suspension culture plates (Greiner Bio-One, Kremsmünster, Austria) 24 h after transfection. Cells were then cultivated with Spheromax CSC Medium (Promocell, Heidelberg, Germany). After 7 days, spheres were counted (number per microscopic field of view, 50× magnification) and the area was measured using an open source ImageJ macro, manually correcting visually incorrect counts [65 (link)].
Suspension culture plates
Manufactured by Greiner
Sourced in Austria
Suspension culture plates are a type of laboratory equipment designed for the cultivation and maintenance of cells in a suspended state. They provide a controlled environment for the growth and proliferation of cells that are not adherent to a surface.
Automatically generated - may contain errors
Lab products found in correlation
Spheromax csc medium, by PromoCell (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (1 mentions)
Mycoalert assay control set, by Lonza (1 mentions)
Cellbanker 2, by AMS Biotechnology (1 mentions)
Matrigel, by Corning (1 mentions)
Pan monocyte isolation kit, by Miltenyi Biotec (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Kx 21n, by Sysmex (1 mentions)
5 protocols using suspension culture plates
1
Sphere Formation Assay Protocol
2
Disruption and Encapsulation of Organoids
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Organoids embedded in BME droplets were disrupted by suspending the content of the wells using a P1000 pipette and transferring it to a 15 ml falcon tube. The volume was adjusted to 15 ml using + / + / + medium and then centrifuged at 400 g for 5 min. Pellets obtained were resuspended in 2 ml TrypLE Express (Life Technologies) and incubated at 37 °C for 5–15 min. The digestion process was monitored under a microscope, and mechanical shearing using a P1000 pipette was performed intermittently until the organoids were disrupted into single cells. The digestion was stopped by topping up the tubes with + / + / + medium, followed by centrifugation (400g, 5 min). After removing the supernatant, the cells were resuspended in 70% BME in + / + / + medium. Organoid density was rechecked under the microscope before plating; if too dense, additional 70% BME in + / + / + was added. Domes of 10–20 µl were plated on pre-heated suspension culture plates (Greiner), inverted, and incubated at 37 °C for 15–20 min for BME solidification. Once solidified, pre-warmed culture medium supplemented with 10 µM Y-27632 was added, and the plates were incubated in a 37 °C, 5% CO2 incubator.
3
Expanding Human Bronchial Epithelial Cells
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Sorted hBEC were grown as organoids by plating onto suspension culture plates (Greiner Bio-One) in hemispheres of growth factor reduced Matrigel (Corning) and grown in hBEC Expansion Medium. For the first three days of culture medium was supplemented with 100 ng/mL Noggin and 100 ng/mL Wnt, as previously described (Huch et al., 2013 (link), 2015 (link)). Medium was changed every three days and cells passaged by mechanical disruption once confluent every 7 to 10 days. Cells were routinely tested for mycoplasma contamination using MycoAlert Assay Control set (Lonza).
For GMP-compliant culture cells were cultured in 2D in 6 well plates covered in 1 mg/mL Laminin 1 (Biolamina).
For long-term storage, organoids were washed twice with Advanced DMEM/F-12 (GIBCO) to remove Matrigel, mixed with cryopreservation solution (CellBanker 2, Amsbio) and stored in cryovials at −80°C for 72 h, then transferred to liquid nitrogen for long term storage. hBEC expansion medium reagents and concentrations are stated inTable S4 .
For GMP-compliant culture cells were cultured in 2D in 6 well plates covered in 1 mg/mL Laminin 1 (Biolamina).
For long-term storage, organoids were washed twice with Advanced DMEM/F-12 (GIBCO) to remove Matrigel, mixed with cryopreservation solution (CellBanker 2, Amsbio) and stored in cryovials at −80°C for 72 h, then transferred to liquid nitrogen for long term storage. hBEC expansion medium reagents and concentrations are stated in
4
Isolation and Characterization of Human Monocytes
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Freshly drawn human whole blood anticoagulated with EDTA was diluted 1:2 in PBS containing 5 mM EDTA, and peripheral blood mononuclear cells (PBMCs) were enriched by density gradient centrifugation on Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden) as previously described52 (link). Monocytes were isolated from PBMCs by negative depletion of non-monocytes (Pan Monocyte Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described42 (link). Their purity was determined by cell counting (Sysmex KX-21N, Sysmex, Neumuenster, Germany) as well as by flow cytometry after staining with anti-CD45-PB and anti-CD14-PE as monocyte markers as well as anti-CD41-PC7 for platelet origin. Monocyte purity was calculated as ratio of CD14+CD45+ cells (monocytes) to all CD45+ cells. Monocyte viability was assessed by trypan blue exclusion. Monocytes were kept in RPMI medium supplemented with 10% EV-depleted AB serum in suspension culture plates (Greiner Bio-One) overnight prior to further use.
5
Growth Assays of S. cerevisiae Strains
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Growth assays of S. cerevisiae were conducted with strain CEN.PK2-1C grown on SC media with sucrose at different concentrations (4%, 1%, 0.25%, 0.0625% (w/v)). The verifications of metabolic inefficiencies in S1 Text , Appendix C, C1 Fig were conducted with an engineered strain of S. cerevisiae (TM6*). This strain has reduced hexose transport capabilities that causes it to have fully respiratory metabolism, which has higher efficiency in terms of ATP yield than the wild-type that has respiro-fermentative metabolism [43 (link)]. Overnight cultures were established in 5 ml SC with 1% (w/v) glucose and grown overnight at 30°C with 180 rpm shaking. Cells were washed and inoculated into fresh SC media containing sucrose at the specified concentrations at an initial density of 500 CFU/μl which was determined based on spectrophotometer measurements calibrated to known densities. Cultures were inoculated into 48-well suspension culture plates (640 μl per well (Greiner Bio-One)) and incubated in a FLUOstar Omega microplate reader (BMG) at 30°C with 700 rpm orbital shaking. Population density was measured by OD 620 nm approximately every 15 minutes and converted to cell density (CFU) based on previously used calibrations [14 (link)].
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