For the spheroid formation assays, Huh7 cells were treated with MCL (30 µM) or DMSO for 8 hr and further incubated with fresh medium for 24 hr. Subsequently, 1000 cells were collected, suspended in DMEM/F12 medium containing 20 ng/mL EGF, 20 ng/mL basic FGF and B27 supplements, and seeded on a Costar® Ultra Low Attachment 6-well plate (Corning) for 14 days. Spheroids were counted to measure the spheroid-forming index when larger than 100 µm. To evaluate the expression of the stemness marker, HCC cells were preformed to fluorescence-activated cell-sorting cytometry (FACS) analysis. After treatment of MCL as above, 1×106 cells were collected, washed, incubated for 30 min at 4 °C with respective fluorescence-conjugated antibodies including CD44-FITC, EpCAM-FITC, and CD133-PE (BD, Biosciences, San Jose, CA, USA). The appropriate fluorochrome-conjugated, isotype-matched antibodies were used as a control to establish background staining. Samples were acquired on a FACS Forte and data were analyzed with DIVA software (BD, Biosciences, San Diego, CA).
Costar ultra low attachment 6 well plate
Manufactured by Corning
Sourced in United States
The Costar ultra-low attachment 6-well plates are designed for cell culture applications that require minimized cell-surface interactions. The plates feature a hydrophilic, non-toxic, and non-pyrogenic surface treatment that prevents cell attachment, enabling the formation of 3D cell aggregates, spheroids, or organoids.
Automatically generated - may contain errors
Lab products found in correlation
Ar009, by R&D Systems (2 mentions)
Pdgf aa 221 aa, by R&D Systems (2 mentions)
Cd44 fitc, by BD (1 mentions)
Epcam fitc, by BD (1 mentions)
Cd133 pe, by BD (1 mentions)
Diva software, by BD (1 mentions)
22 g needle, by Thermo Fisher Scientific (1 mentions)
Nutristem xf, by Sartorius (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
Neural basal, by Thermo Fisher Scientific (1 mentions)
Bone morphogenetic protein 4 (bmp4), by Merck Group (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Human bfgf 2, by Thermo Fisher Scientific (1 mentions)
Dasatinib, by Selleck Chemicals (1 mentions)
Psgro hesc ipsc growth medium, by System Biosciences (1 mentions)
Pe annexin 5 apoptosis detection kit with 7 aad, by BioLegend (1 mentions)
13 protocols using costar ultra low attachment 6 well plate
1
Stemness Evaluation of Huh7 Cells
2
Collagen-Coated Bead Adhesion Assay
3
Tumorsphere formation and characterization
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HCC tumorspheres were generated from scrambled shRNA or shMALAT1 SK-Hep1 and HepG2 single-cell suspension. SK-Hep1 or HepG2 cells were seeded at a density of 5 × 104 per well into Corning® Costar® ultra-low attachment 6-well plates (Corning, NY, USA) containing growth factors for CSCs enrichment stem cell medium (Nutristem-XF, Biological Industries, Israel) and incubated at 37 °C in humidified 5% CO2 incubator for 5–7 days, followed by visualization of the primary tumorspheres (diameter ≥100 μm) under an inverted microscope. Secondary tumorspheres were generated by dissociating the primary tumorspheres by trypsinization, pipetting dissociated primary tumorspheres through a 22G needle (Thermo Fisher Scientific Inc., Bartlesville, OK, USA) to obtain single-cell suspension. After dissociation of the primary tumorspheres, cells were seeded as earlier described for primary tumorspheres. After 5–7 days of culture, secondary orospheres consisting of ≥100 µm were counted, and images taken under microscope. Tumorsphere size, quantity and formation efficiency were evaluated. Tumorsphere formation efficiency (TFE) was evaluated using the formula: TFE = (number of sphere formed/number of single cells plated) × 100.
4
Differentiation of iPSCs to OPCs
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iPSCs were differentiated to OPCs as previously described43 (link),44 (link). In brief, iPSCs were isolated from their iMEF feeder
layer using 1.5mg/mL collagenase type IV (17104019, ThermoFisher) and
dissociated with either 0.25% Typsin-EDTA or Accutase and seeded at
7.8×104 cells/cm2 on Costar Ultra-Low attachment 6-well
plates (3471, Corning). Cultures were then directed through a stepwise
differentiation process to generate pure populations of OPCs. OPCs were
maintained in “OPC medium” consisting of DMEM/F12 (11320082,
ThermoFisher), 1x N2 supplement (AR009, R&D Systems), 1x B-27 without
vitamin A supplement (12587–010, ThermoFisher), and 1x Glutamax
(collectively “N2B27 medium”), supplemented with 20 ng/mL
fibroblast growth factor 2 (FGF2; 233-FB, R&D Systems) and 20 ng/mL
platelet-derived growth factor-AA (PDGF-AA; 221-AA, R&D Systems). Medium was
changed every other day. All cell cultures in the laboratory are routinely
tested for mycoplasma contamination with consistently negative results. For
characterization of purity, iPSC-derived OPCs were fixed with 4% PFA and
immunostained for canonical OPC transcription factors, OLIG2 and SOX10, and
counterstained with DAPI.
layer using 1.5mg/mL collagenase type IV (17104019, ThermoFisher) and
dissociated with either 0.25% Typsin-EDTA or Accutase and seeded at
7.8×104 cells/cm2 on Costar Ultra-Low attachment 6-well
plates (3471, Corning). Cultures were then directed through a stepwise
differentiation process to generate pure populations of OPCs. OPCs were
maintained in “OPC medium” consisting of DMEM/F12 (11320082,
ThermoFisher), 1x N2 supplement (AR009, R&D Systems), 1x B-27 without
vitamin A supplement (12587–010, ThermoFisher), and 1x Glutamax
(collectively “N2B27 medium”), supplemented with 20 ng/mL
fibroblast growth factor 2 (FGF2; 233-FB, R&D Systems) and 20 ng/mL
platelet-derived growth factor-AA (PDGF-AA; 221-AA, R&D Systems). Medium was
changed every other day. All cell cultures in the laboratory are routinely
tested for mycoplasma contamination with consistently negative results. For
characterization of purity, iPSC-derived OPCs were fixed with 4% PFA and
immunostained for canonical OPC transcription factors, OLIG2 and SOX10, and
counterstained with DAPI.
5
Differentiation of iPSCs to OPCs
6
Maintaining Pancreatic Cancer Tumorspheres
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Established pancreatic cancer tumorspheres [10] (link) were maintained in sphere media as described previously [19] (link), [20] (link) with modifications [50% NeuralBasal (Invitrogen, Carlsbad, CA), 1% N2 Supplement (Invitrogen, Carlsbad, CA), 2% B27 supplement (Invitrogen, Carlsbad, CA), 1% Antibiotic-Antimycotic (Invitrogen, Carlsbad, CA), 10 ng/mL BMP4 (Sigma), 10 ng/mL LIF (Sigma), 20 ng/mL human bFGF-2 (Invitrogen, Carlsbad, CA), all in 1∶1 DMEM/F12 (Invitrogen, Carlsbad, CA)]. Tumorspheres were passaged every 6 days. For passaging, tumorspheres were dissociated with 0.05% trypsin for 2–5 min and then immediately washed twice with 40 mL PBS. Cells were then passed through a 40 μm nylon mesh cell strainer, counted and plated in fresh sphere medium in Costar ultra low-attachment 6 well plates (Corning, Lowell, MA).
7
Anoikis-Resistant Cell Selection
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The adherent cells were trypsinized (A610681-0100, Sangon Biotech Co., Ltd., Shanghai, China) so as to obtain the single cell suspension and then seeded at 106 per well in Costar ultra-low attachment 6-well plates (3471, Corning Incorporation, Corning, NY, USA). Poly-hydroxyethyl methacrylate (poly-HEMA, ST1582, Beyotime Biotechnology, Shanghai, China) was pre-dissolved in 95% ethanol (493511, Sigma-Aldrich, Ontario, Canada), and the solution was added into the culture plates to induce the anoikis in the cell. The cells were passaged every three days. After continuous passages, the anoikis resistant cells were selected and collected as AR group for biological characteristics analysis. Cells under adherent culture were used as the control group and received no treatment.
8
Sphere Formation Assay for Cancer Stem Cells
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Sphere-forming assay was performed as previously described [13 (link), 14 (link)]. Briefly, C9IV3 and HSC3 cells were transfected with plasmids containing siCon or siKRT17 for 24 h, or miR-Con, miR-485-5p, miR-485-5p + KRT17 for 48 h, or in combination with 1 μM dasatinib (Selleckchem, Houston, Texas, USA) treatment for 48 h prior to sphere forming assay. Transfected and/or treated cells were cultured in stem cell selective medium at cell density of 1 × 105 cells per well with 3 ml of PSGro hESC/iPSC growth medium (System Biosciences, Palo Alto, CA, USA) in Costar ultra-low attachment 6-well plates (Corning, Oneonta, NY, USA). The total numbers of spheres formed were counted under microscope at day 10.
9
Transplantation of Kidney Organoids
10
Culturing and Passaging BR-luc Cells
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BR-luc cells were seeded at a density of 1 × 106 cells per well in Costar ultra-low attachment 6-well plates (Corning). The cells were incubated with 3 ml DMEM media in 5% CO2 at 37 °C. After 2 days, culture media were collected in 15 ml conical tubes and cells were precipitated at 1000 rpm for 0.5 minutes. After two rounds of washing with phosphate buffered saline (PBS), large cell aggregates were separated into small aggregates by multiple pipetting through a 1 ml pipette tip.
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