All work with human material was performed with IRB approval. fRPCs were isolated from human fetal neural retina at 16 weeks gestational age as previously described (Luo et al., 2014 (link)). Whole neuroretina was separated from the RPE layer, minced, and digested with collagenase I (Sigma-Aldrich). Cells and cell clusters were plated onto human fibronectin (Akron)-coated flasks (Nunclon Delta) in Ultraculture Media (Lonza), supplemented with 2 mM L-glutamine (Invitrogen), 10 ng/ml rhbFGF (Peprotech), and 20 ng/ml rhEGF (Peprotech) in a low-oxygen incubator (37°C, 3% O2, 5% CO2, 100% humidity). Cells were passaged at 80% confluency using TrypZean (Sigma-Aldrich), benzonase (EMD Chemicals), and Defined Trypsin Inhibitor (Invitrogen).
Ultraculture media
Manufactured by Lonza
Sourced in United States
Ultraculture Media is a product designed for cell culture applications. It provides a standardized and optimized growth medium for the cultivation of various cell lines. The media composition is formulated to support the proliferation and maintenance of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (2 mentions)
Phenol red free dmem with 10 fbs, by Thermo Fisher Scientific (2 mentions)
Amicon pro affinity concentration kit protein a with 10kda amicon ultra 0.5 device, by Merck Group (2 mentions)
293t cell, by American Type Culture Collection (2 mentions)
Rh bfgf, by Thermo Fisher Scientific (1 mentions)
Trypzean, by Merck Group (1 mentions)
Defined trypsin inhibitor, by Thermo Fisher Scientific (1 mentions)
Rhegf, by Thermo Fisher Scientific (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
Pmd2 g envelope plasmid, by Addgene (1 mentions)
Polybrene, by Merck Group (1 mentions)
Glucose and l glutamine, by Lonza (1 mentions)
Hek293t, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Anti trα β, by Santa Cruz Biotechnology (1 mentions)
Glucose and glutamax, by Thermo Fisher Scientific (1 mentions)
Sh sy5y, by American Type Culture Collection (1 mentions)
Anti e cadherin, by Santa Cruz Biotechnology (1 mentions)
Sh sy5y, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
8 protocols using ultraculture media
1
Isolation and Culture of Human fRPCs
2
Lentiviral Transduction of Keratinocytes
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Lentiviral NLS-iCre-GFP was a gift from Elaine Fuchs (The Rockefeller University). Lentivirus was produced as previously described (Bhattarai et al., 2019 ). Briefly, the lentiviral plasmid was cotransfected with a pMD.2G envelope plasmid (Addgene #12259) and a psPAX2 packaging plasmid (Addgene #12260) using calcium phosphate/HBS into 293FT cells cultured in DMEM containing 10% fetal bovine serum (FBS) and G418/Geneticin. One day following transfection the media was replaced with Ultraculture Media (Lonza 12-725F) supplemented with 1% penicillin/streptomycin/l -glutamine, 1 mM sodium pyruvate, 0.075% sodium bicarbonate, and 5 mM sodium butyrate. Forty-eight hours after transfection, viral supernatant was collected from the cultures.
Transduction of keratinocyte cultures was performed as previously described (Bhattarai et al., 2019 ). Briefly, keratinocytes were grown to 70–80% confluency in a six-well plate. Immediately prior to viral transduction, 3 μl of Polybrene (10 mg/ml; Sigma Aldrich 107689) and 50–100 μl of viral supernatant were added per well. The plate was centrifuged at 1100 × g at 37°C for 30 min, after which the media was replaced with fresh low-calcium E-media. Successful transduction was confirmed 2–3 d afterward.
Transduction of keratinocyte cultures was performed as previously described (Bhattarai et al., 2019 ). Briefly, keratinocytes were grown to 70–80% confluency in a six-well plate. Immediately prior to viral transduction, 3 μl of Polybrene (10 mg/ml; Sigma Aldrich 107689) and 50–100 μl of viral supernatant were added per well. The plate was centrifuged at 1100 × g at 37°C for 30 min, after which the media was replaced with fresh low-calcium E-media. Successful transduction was confirmed 2–3 d afterward.
3
Cell Line Characterization and Culture Protocol
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Cell lines used in this study were: MMQ (ATCC, CRL-10609), GC [51] (link), GHFT [52] (link), HEK293T (ATCC, CRL-11268) and SH-SY5Y (ATCC, CRL-2266). Antibodies used in this work included: anti-GH, anti-Prl (prolactin), anti-TSHβ, anti-Pit-1 (1769), anti-TRα/β (Santa Cruz, sc-772, fl-408), anti-FLAG (Sigma, Cat. # F3165), anti-E-cadherin (Santa Cruz, sc-7870, H-108) and anti-SmcHD1 antibody (Abcam, Cat. # ab31865). 5-Azacytidine was purchased from Invitrogen.
MMQ cells from rat pituitary were cultured in Ultraculture media (Biowhittaker, Cat. #12-725F) without L-glutamine and supplemented with 5% fetal bovine serum (FBS) and were maintained in a humidified atmosphere containing 5% CO2 at 37°C. HEK293T, SH-SY5Y and GHFT cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 4.5 g/L Glucose and L-Glutamine (Biowhittaker, Cat. # 12-604F) and supplemented with 10% fetal bovine serum (FBS). GC cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 4.5 g/L Glucose and Glutamax (Invitrogen, Cat. # 10566-016) supplemented with 12.5% horse serum and 2.5% FBS. Where indicated, cells were treated with 0.25% 5-azaC every 24 hrs for a minimum of 72 hrs.
MMQ cells from rat pituitary were cultured in Ultraculture media (Biowhittaker, Cat. #12-725F) without L-glutamine and supplemented with 5% fetal bovine serum (FBS) and were maintained in a humidified atmosphere containing 5% CO2 at 37°C. HEK293T, SH-SY5Y and GHFT cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 4.5 g/L Glucose and L-Glutamine (Biowhittaker, Cat. # 12-604F) and supplemented with 10% fetal bovine serum (FBS). GC cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) containing 4.5 g/L Glucose and Glutamax (Invitrogen, Cat. # 10566-016) supplemented with 12.5% horse serum and 2.5% FBS. Where indicated, cells were treated with 0.25% 5-azaC every 24 hrs for a minimum of 72 hrs.
4
Cytotoxicity Assessment of Cigarette and E-Cig Aerosols
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The cytotoxicity of WA was assessed as previously described [22 (link)]. In brief, NCI-H292 human bronchial epithelial cells (American Type Culture Collection, Middlesex, UK) were grown on cell-culture inserts, and transitioned to the air–liquid interface (ALI) immediately prior to exposure. Cells were exposed directly to undiluted 3R4F WA for up to 7 puffs and undiluted e-cig aerosols for up to 1000 puffs. As an air control, cultures were exposed to air in an exposure chamber at the same frequency and volume as WA exposure. ALI and submerged cell control cultures were kept in an incubator during the exposure period.
Following exposure, cell culture inserts were transferred to 12-well culture plates. Supplemented UltraCULTURE™ media (Lonza, Walkersville, MD, USA) were added to the basal and apical compartments, and the cells were incubated for a further 24 h. Each exposure was conducted 6–8 times with 3 culture inserts per experiment. Cell viability was measured by the NRU assay 24 h after aerosol exposure, as described previously [34 ].
Following exposure, cell culture inserts were transferred to 12-well culture plates. Supplemented UltraCULTURE™ media (Lonza, Walkersville, MD, USA) were added to the basal and apical compartments, and the cells were incubated for a further 24 h. Each exposure was conducted 6–8 times with 3 culture inserts per experiment. Cell viability was measured by the NRU assay 24 h after aerosol exposure, as described previously [34 ].
5
Transient Transfection of 293T Cells
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HCAbs were generated by transient transfection of 293T cells (American Type Culture Collection, ATCC). Briefly, 4 × 106 293T cells were seeded into Poly-L-Lysine coated 10 cm dishes one day before transfection, in DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin. The next day, 25 μg of expression plasmids pVAX-J3 or pVAX-A6 were introduced into cells using calcium phosphate transfection. After overnight incubation, cells were washed with PBS and media was replaced with serum-free UltraCULTURE media (Lonza) or phenol red-free DMEM with 10% FBS (Thermo-Fisher). Supernatants were harvested after 3 days, clarified by centrifugation for 15 mins at 2500 × g and filtered using a 0.45 μm filter. Antibodies were purified using Amicon Pro Affinity Concentration Kit Protein A with 10kDa Amicon Ultra-0.5 Device (Millipore-Sigma). Antibody concentrations were determined using an IgG ELISA, described below.
6
Isolation and Expansion of Human Retinal Progenitor Cells
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hRPCs isolated from donated neural retinas of 16 to 18 weeks gestational age were obtained from the Eye Bank of He Eye Hospital in Shenyang, China. The donor signed the informed consent form. All works with human samples were performed after obtaining approval from the Institutional Review Board (IRB) of He Eye Hospital of He University (approval K007.01, March 2019). The protocol used a previously reported approach [13 (link)]. Briefly, the whole neuroretina was taken, then minced and digested with TrypLE express (Invitrogen) for 2 min. The cell pellet was resuspended using Ultraculture media (Lonza), supplemented with 1% N2 neural supplement, L-glutamine (2 mM), penicillin-streptomycin (100 U/ml), and recombinant human epidermal growth factor (EGF, 20 ng/ml) and basic fibroblast growth factor (bFGF, 20 ng/ml) (all purchased from Invitrogen), followed by inoculation in fibronectin pre-coated cell flasks in an incubator (37°C, 5% CO2). The hRPCs were passaged when the confluence reached 80%. The hRPCs at passage 6 were used for the experiments.
7
Generation of Recombinant Antibodies from 293T Cells
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HCAbs were generated by transient transfection of 293T cells (American Type Culture Collection, ATCC). Briefly, 4 x 106 293T cells were seeded into Poly-L-Lysine coated 10 cm dishes one day before transfection, in DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin. The next day, 25 μg of expression plasmids pVAX-J3 or pVAX-A6 were introduced into cells using calcium phosphate transfection. After overnight incubation, cells were washed with PBS and media was replaced with serum-free UltraCULTURE media (Lonza) or phenol red-free DMEM with 10% FBS (Thermo-Fisher). Supernatants were harvested after 3 days, clarified by centrifugation for 15 mins at 2500 x g and filtered using a 0.45 μm filter. Antibodies were purified using Amicon Pro Affinity Concentration Kit Protein A with 10kDa Amicon Ultra-0.5 Device (Millipore-Sigma). Antibody concentrations were determined using an IgG ELISA, described below.
8
Mouse DRG Neuron Isolation and Culture
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