For 2D culture, three replicate cultures at 70-80% confluence for each ZMEL1-PRO and -INV were utilized. For 3D culture, three replicate cultures for each ZMEL1-PRO and -INV grown in Corning Ultra-Low Attachment Surface 6-well plates (Corning #3471) for 48 hours were utilized. One individual replicate of RNA-seq of ZMEL1-PRO grown in 3D culture was excluded due to low RIN score, poor SeQC metrics and poor clustering with other replicate samples. For tfap2a/e CRISPR, three independent batches of ZMEL1-PRO cells nucleofected with either sg_scr or sg_tfap2a/e and grown in 2D conditions were utilized at 8-16 days post nucleofection.
Ultra low attachment surface 6 well plate
Manufactured by Corning
Sourced in United States
The Corning Ultra-Low Attachment Surface 6-well plates are designed for cell culture applications that require minimal cell attachment to the surface. The plates feature a hydrophilic, non-adherent coating that promotes the formation of spheroids, suspension cultures, and other 3D cell models.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Srx 101a, by Konica Minolta (2 mentions)
Clone d69c12, by Cell Signaling Technology (2 mentions)
Clone 12d1, by Cell Signaling Technology (2 mentions)
Clone 9f3, by Cell Signaling Technology (2 mentions)
Immobilon p transfer membrane, by Merck Group (2 mentions)
Amicon ultra, by Merck Group (2 mentions)
Epidermal growth factor (egf), by Merck Group (2 mentions)
Basic fibroblast growth factor (bfgf), by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Ix70 inverted microscope, by Hamamatsu Photonics (1 mentions)
Orca er camera, by Hamamatsu Photonics (1 mentions)
Human transferrin, by Roche (1 mentions)
Human insulin, by Roche (1 mentions)
Basic fibroblast growth factor (bfgf), by Fujifilm (1 mentions)
Bovine serum albumin (bsa), by Nacalai Tesque (1 mentions)
Bz x viewer, by Keyence (1 mentions)
15 protocols using ultra low attachment surface 6 well plate
1
3D and 2D Culture of ZMEL1 Cells
2
Efficient iPSC Differentiation Protocol
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Differentiation was carried out as previously described48 with slight modifications. Briefly, iPSCs were passaged sparsely to 6‐well Matrigel‐coated plates and cultured for 8–10 days in mTeSR1 with daily medium changes until the colonies were approximately 5 mm in diameter. Next, embryoid bodies (EBs) were formed by gently lifting colonies using a cell lifter. Colonies were gently transferred to a 15 mL conical tube using a 10 mL serological pipette. Colonies were allowed to gravity settle for approximately 5 min and supernatant was aspirated. Cells were gently resuspended in mTeSR1 supplemented with 10 μM ROCK inhibitor (Y‐27632) and transferred to Ultra‐Low Attachment Surface 6‐well plates (Corning) containing 4 mL medium per well. EBs were allowed to form for 4 days with a partial (2/3) medium change after 48 h. On day 4, EBs were collected, washed, resuspended in X‐VIVO15 supplemented with GlutaMAX™ (Gibco) and 2‐mercaptoethanol (Gibco), and transferred to adherent, cell culture‐treated plates.
3
Quantifying Anchorage-Independent Growth
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The ability of anchorage-independent growth was quantified as clonogenicity in 15% methylcellulose-containing full growth medium [28 (link)]. Cells were harvested by routine trypsinisation, dissolved in 15% methylcellulose-containing full growth medium and partitioned in triplicates onto ultra-low attachment surface 6-well plates (Corning); 20,000 cells per well were used. Colonies were counted after four weeks of culture, with supplementation with the normal growth medium every week. Photographic documentation was taken by the Olympus IX 70 inverted microscope equipped with the Hamamatsu Orca-ER camera. Experiments were performed in triplicates and repeated four times with similar results.
4
Tumor Spheroid Formation Assay
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Cells (1 × 105 cells/well) at 10 days after retroviral transduction were seeded to Ultra-Low Attachment Surface 6 well plates (Corning Inc., Corning, NY, USA) in a serum-free DMEM medium containing 10 ng/mL basic fibroblast growth factor (bFGF; Wako, Osaka, Japan), 10 mg/mL human insulin (CSTI, Miyagi, Japan), 100 mg/mL human transferrin (Roche, Basel, Switzerland), and 100 mg/mL bovine serum albumin (BSA; Nacalai Tesque) and incubated at 37 °C in a 5% CO2 incubator for 10 days [13 (link)]. Tumor spheroids were manually counted under an inverted phase contrast microscope (BZ-X710 Microscope and BZ-X Viewer, BZ-X Analyzer imaging system, Keyence, Osaka, Japan). All morphometric studies were performed by two examiners blinded to treatment conditions.
5
Western Blot Analysis of Laminin-α4 and Cell Cycle Proteins
6
Sarcosphere Formation and Side Population
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The ability to grow in sarcospheres was assessed as described [29 (link),30 (link)]. Briefly, cells were harvested by routine trypsinisation at ~80% confluence. Then, 100,000 cells were seeded per one well in ultra-low attachment surface 6-well plates (Corning, NY, USA) in serum-free DMEM/1% methylcellulose medium supplemented with the recombinant mouse FGF2 (Sigma SRP4038–10 ng/mL, Sigma, Prague, CZ) EGF from murine submaxillary gland (Sigma E4127–10 ng/mL, Sigma, Prague, CZ) and 1× N2 supplement (Gibco/Invitrogen), with regular addition of FGF-2 and EGF every other day. Following 10–14 days in culture, colonies that contained >10 cells were quantitated by inverted phase contrast microscopy. Experiments were performed in triplicates and repeated a minimum of twice with similar results. The side population was analysed as DyeCycle™ Violet (ThermoFischer Scientific, Carslbad, CA, USA) dim cells, as described previously [31 (link)].
7
Differentiation of iPSCs to Embryoid Bodies
8
Western Blot Analysis of Laminin-α4 and Cell Cycle Proteins
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For detection of laminin-α4, conditioned media was prepared by concentrating serum-free supernatant obtained from cultured cancer cells incubated for 24hr using 100k cutoff Amicon Ultra (Millipore) centrifuge tubes. For detection of p27, p21, and β-tubulin, cell lysates were collected from cells that had been cultured overnight in Ultra-Low Attachment Surface 6-well plates (Corning). Protein from conditioned media or cell lysates were separated using SDS–polyacrylamide gel electrophoresis, transferred to Immobilon-P Transfer Membrane (Millipore), and probed with antibodies against human laminin-α4 (1:200; clone 6C3, sc-130541, Santa Cruz Biotechnology) or p27 (1:1,000; clone D69C12, Cell Signaling Technology), p21 (1:1,000; clone 12D1, Cell Signaling Technology), or β-tubulin (1:5,000; clone 9F3, Cell Signaling Technology). Primary antibody was chemiluminescently detected using horseradish peroxidase–conjugated secondary antibody (1:10,000), ECL 2 Western Blotting Substrate (Pierce) and the SRX-101A (Konica Minolta) developer. Quantification of western blots was performed using ImageJ (NIH) software. p27 signal intensity was normalized to β-tubulin signal intensity.
9
Tumor Spheroid Formation Assay
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HCT116 spheroid cells transfected with empty vector or SNORD1C-pcDNA and SW480 spheroid cells transfected with sh-nc or sh-SNORD1C were seeded onto Ultra-Low Attachment Surface 6-well plates (Corning Inc.) with a density of 2000 cells/well in serum-free DMEM-F12 supplemented with 10 ng/mL bFGF, 20 ng/mL EGF (Sigma-Aldrich, St. Louis, MO, USA), and 2% B27 (Invitrogen). The medium was changed every 3–4 days during sphere formation. After culture for 10–14 days, under an inverted microscope, the number of tumorspheres was counted, and spheres >50 μm in diameter were calculated and plotted.
10
Tumor Sphere Formation Assay
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A total of 5×103 BCa cells were seeded into ultra-low attachment surface 6-well plates (Corning, Inc.) and maintained in DMEM with 20 ng/ml epidermal growth factor (PeproTech, Inc.), 20 ng/ml basic fibroblast growth factor (PeproTech, Inc.) and 2% B27 (Thermo Fisher Scientific, Inc.). After 1 week of cultivation, the number of tumor spheres was counted in five independent fields (21 (link)). The experiments were repeated three times.
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