Cells were seeded in a 384-well CellBIND (Corning) at 1,000 cells/well with phenol red-free growth media and incubated overnight (~12-14h) at 37°C, 5% CO 2 . The cells were then treated with drug treatments at the concentrations listed as well as Caspase 3/7 Green Apoptosis Assay Reagent (Sartorius) at a 1:1000 dilution (n=4 wells/treatment). Cells were imaged using the Incucyte S3 live imaging system (Sartorius). Phase and green contrast images were captured every 4h for 72h. Images were analyzed using the integrated software. Primary human VS cells were seeded in 384-well CellBIND plates (Corning) at 1,000 cells/well with Schwann media and incubated overnight at 37°C and 5% CO 2 . Cells were then treated with 0.05% dimethyl sulfoxide (DMSO; control condition), omipalisib (2, 4, 8nM), dasatinib (200, 400, 800nM), or the combination in maintenance media consisting of Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Seradigm) and 1% penicillin-streptomycin (n=6 per condition). At 96 hrs, the ApoTox-Glo (Promega) assay was used to detect live cell protease activity for viability and cleaved caspase-3/7 activity for apoptosis using the Glomax Discover System (Promega).
Cellbind
Manufactured by Corning
Sourced in United States
CellBIND is a proprietary surface treatment for cell culture vessels that enhances cell attachment and growth. It provides a consistent and reliable surface for a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Cytoflex, by Beckman Coulter (2 mentions)
Cytexpert software, by Beckman Coulter (2 mentions)
Ficoll hypaque, by GE Healthcare (2 mentions)
Steadylite plus luciferase reagent, by PerkinElmer (2 mentions)
Pgl4.32 nf κb luciferase, by Promega (2 mentions)
Flipr tetra, by Molecular Devices (2 mentions)
Caspase 3 7 green apoptosis assay reagent, by Sartorius (1 mentions)
Incucyte s3 live imaging system, by Sartorius (1 mentions)
Apotox glo, by Promega (1 mentions)
Glomax discover system, by Promega (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Hek293, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Merck Group (1 mentions)
K sfm kit, by Thermo Fisher Scientific (1 mentions)
Live dead violet stain, by Thermo Fisher Scientific (1 mentions)
Click it edu kit, by Thermo Fisher Scientific (1 mentions)
Fxcycle stain, by Thermo Fisher Scientific (1 mentions)
Fluo 4 nw calcium assay kit, by Thermo Fisher Scientific (1 mentions)
52 protocols using cellbind
1
Apoptosis Assay of NF1 Tumor Cell Lines
2
Prostate Cell Culture Protocols
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Normal human epithelial prostate cells (RWPE-1), which express the androgen receptor, were compared with androgen sensitive, human prostate cancer cells (LNCaP). Androgen independent human prostate cancer cells (PC3) were used for in vitro scratch assays due to their ability to produce a monolayer in culture. Human embryonic kidney cells (HEK293) were used for tumor necrosis factor-α (TNF-α) shedding experiments.
All cells were purchased from the American Type Culture Collection (Manassas, VA, USA). HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) [low glucose; Gibco] containing 10 % fetal calf serum (FCS) and 1 % penicillin/streptomycin (Invitrogen, USA). RWPE-1 cells were cultured in Keratinocyte Serum Free Medium (K-SFM; GIBCO) containing 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml human recombinant epidermal growth factor (EGF) provided with the K-SFM kit. LNCaP and PC3 cells were cultured in Roswell Park Memorial Institute-1640 media (RPMI) (Sigma-Aldrich, Germany) with 10 % FCS and 1 % penicillin/streptomycin. To maintain viable healthy and undifferentiated cells, RWPE-1, LNCaP and PC3 cells were maintained until they reached 70 % confluency and were then transferred into a 75 cm2 flask. Cells were split into 6 well Cell Bind (Costar), 12 well Cell Bind (Costar) or 96 well cell culture plates for further studies.
All cells were purchased from the American Type Culture Collection (Manassas, VA, USA). HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) [low glucose; Gibco] containing 10 % fetal calf serum (FCS) and 1 % penicillin/streptomycin (Invitrogen, USA). RWPE-1 cells were cultured in Keratinocyte Serum Free Medium (K-SFM; GIBCO) containing 0.05 mg/ml bovine pituitary extract (BPE) and 5 ng/ml human recombinant epidermal growth factor (EGF) provided with the K-SFM kit. LNCaP and PC3 cells were cultured in Roswell Park Memorial Institute-1640 media (RPMI) (Sigma-Aldrich, Germany) with 10 % FCS and 1 % penicillin/streptomycin. To maintain viable healthy and undifferentiated cells, RWPE-1, LNCaP and PC3 cells were maintained until they reached 70 % confluency and were then transferred into a 75 cm2 flask. Cells were split into 6 well Cell Bind (Costar), 12 well Cell Bind (Costar) or 96 well cell culture plates for further studies.
3
Cell Proliferation Assay with EdU
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HS01 cells were seeded at 250,000 cells/well in 6-well CellBIND (Corning) plates, grown to ~80% confluency, and treated with CUDC907 for 24 h. EdU (10 μM; Click-iT EdU kit; Molecular Probes, ThermoFisher) was added during the last 3 h. Cells were harvested with 0.05% trypsin, stained with violet live/dead stain (ThermoFisher), and permeabilized. DNA labeling with FxCycle stain (ThermoFisher) was conducted according to manufacturer’s protocol. Cell populations were identified by flow cytometry on CytoFlex (Beckman Coulter) and analyzed by CytExpert software (Beckman Coulter).
4
Differentiation of NT2.N/A Co-Cultures
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Production of the NT2.N/A co-cultures was based on previous plating methods ([19 (link), 31 (link)] with modifications as previously described by ourselves [32 (link),33 (link)]. Briefly, NT2.D1 cells (2 × 106 cells/75 cm2 flask) were treated (unpassaged, to allow a build-up of layers of cells) for 4 weeks using DMEM high glucose medium (DMEM-HG), changed twice weekly and supplemented with 10% (v/v) foetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin and 1 × 10-5 M all-trans retinoic acid (RA). Differentiated cells were replated (sub-cultured 1:3; replate #1) and incubated for two days, then dislodged and replated onto CellBIND (Corning, Netherlands) 6-well plates (2.5 × 106 cells/well, replate #2) and treated with mitotic inhibitors for 28 days; 0.1 μM cytosine arabinoside (for the first 7 days only), 3 μM fluorodeoxyuridine and 5 μM uridine. No further passaging took place as cells had reached a post-mitotic stage. For some experiments, 12 mm poly-D-lysine (PDL)/Laminin Corning Biocoat round coverslips (VWR International, Lutterworth, UK) were placed in the wells prior to replate #2.
5
Fluorometric Calcium Assay for TP Cells
6
Quantifying Apoptosis in Human and Mouse Cells
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Human (1,000 cells/well) and mouse (2,000 cells/well) MD-SCs were seeded in phenol red-free growth medium in four replicate wells/condition of a 384-well CellBIND (Corning) plate. After ~13 h, drugs (diluted in DMSO) at three concentrations (the IG50 and lower and higher concentrations) and Incucyte® Caspase-3/7 Green Apoptosis Assay Reagent (Sartorius) were added to wells. Wells were imaged using the Incucyte® S3 Live-Cell Analysis system (Sartorius) for 72 h. Phase and green fluorescent images were collected every 4 h and analyzed using the integrated basic analyzer software.
Primary human VS cells were treated with CUDC907 as described for viability assays. The Caspase-Glo® 3/7 Assay (Promega) was used to detect cleaved caspase-3/7 at 36 and 48 h, and the RealTime-Glo™ Annexin V Apoptosis Assay (Promega) was performed to detect phosphatidylserine over 48 h, following manufacturer’s protocols.
Primary human VS cells were treated with CUDC907 as described for viability assays. The Caspase-Glo® 3/7 Assay (Promega) was used to detect cleaved caspase-3/7 at 36 and 48 h, and the RealTime-Glo™ Annexin V Apoptosis Assay (Promega) was performed to detect phosphatidylserine over 48 h, following manufacturer’s protocols.
7
Cytotoxicity Assay for Doxorubicin and SipA
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Cytotoxicity assays were performed as described previously45 (link) with minor modifications. HCT8 cells (2,500 per well) were plated in 96-well Cellbind plates (Corning, Tewksbury, MA) in 100 μl of growth media. After overnight attachment, cells were incubated for 72 h with doxorubicin (1 μg ml−1) and purified SipA (100 μg ml−1), alone and in combination. Verapamil (20 μM) was used as a positive control, alone and in combination with doxorubicin. After treatment, the number of viable cells was determined by a colorimetric cell proliferation assay (CellTiter96 Aqueous One solution; Promega, Madison, WI) according to the manufacturer’s instructions. All studies were conducted in triplicate and performed at least three times independently.
8
Culturing Primary Human Endothelial Cells
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Primary human umbilical vein endothelial cells (HUVEC) were purchased from PromoCell (Heidelberg, Germany) and cultured in a complete endothelial cell growth medium (designated as complete EGM) containing 2% FBS and growth factors supplied by the company’s (cat # C-22010 ready to use kit). HUVEC (passage 2–4) were cultured and maintained on 10 cm culture plates (Cell-bind, Corning) until confluent and grew under standard cell culture conditions (37 °C, 5% CO2). Cells were split into the desired plate one day before the experiments. For Seahorse experiments, cells are attached for at least 24 h before experiments.
9
Generation of HEK 293 CFTR-G542X Stable Cell Line
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HEK 293 cells were plated in a six-well plate (CellBind, Corning, Corning, NY) at 350,000 cells per well. The following day, cells were transfected with 2 µg of CFTR-G542X minigene plasmid, described above, and with 10 µL Lipofectamine 2000 reagent, according to the manufacturer’s protocol (Life Technologies). Transfection media were removed from cells the next day and replaced with DMEM growth media supplemented with 10% bovine serum albumin and 1% penicillin/streptomycin for an additional 24 h. At this time, cells were subjected to selection media containing 800 µg/mL hygromycin for 7 d. Hygromycin-resistant cells were pooled from all wells and expanded for an additional 14 d. The HEK 293 CFTR-G542X stable cell line was shown to express G542X-CFTR protein by Western blots following incubation with a known aminoglycoside readthrough agent (G418)15 (link) (Fig. 1B ). The cell line was shown to express functional CFTR following G418 incubation in an FMP membrane potential assay in comparison with the parental HEK 293 cells to establish G542X-CFTR responsiveness to readthrough modulators.
10
Primary Hepatocyte Isolation and Cultivation
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Primary hepatocytes were isolated from 8-12-week old mice by collagenase perfusion as described previously65 (link). Cells were plated on 12-well or 24-well tissue culture dishes (Cellbind, Corning) and cultured in M199 medium with 10% FBS for 4-6 h before transfection or infection. Before insulin (100 nM) or glucagon (100 nM) stimulation, cells were fasted in serum-free M199 medium for 14 h.
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