Primary mouse RPE cells and retinal microglial cells were prepared from 2-week-old mice based on previously published methods (19 (link), 20 (link)). Enucleated eyes were incubated with 2% dispase (Invitrogen) in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) for 1 h at 37 °C, and neural retinas and eyecups were separated under a surgical microscope (ILLUMIN-i, Endure Medical, Cumming, GA). The RPE layer was peeled from eye cups, and cultured in DMEM containing minimal essential medium non-essential amino acids (Invitrogen), penicillin/streptomycin (Invitrogen), 20 mM HEPES, pH 7.0, and 10% fetal bovine serum. To enrich microglial cells, neural retinas were homogenized and cultured in DMEM containing minimal essential medium non-essential amino acids (Invitrogen), penicillin/streptomycin (Invitrogen), 20 mM HEPES, pH 7.0, and 10% fetal bovine serum for 7 days at 37 °C. Adherent cells to the plastic surface were treated with 0.05% trypsin (Invitrogen), and less adhesive cells were collected as microglial cells. Human primary RPE cells were purchased from Lonza (Walkersville, MD).
Minimal essential medium nonessential amino acids
Manufactured by Thermo Fisher Scientific
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Minimal essential medium nonessential amino acids is a cell culture medium component that provides a balanced mixture of essential and nonessential amino acids to support the growth and maintenance of various cell types in vitro.
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Lab products found in correlation
Sodium pyruvate, by Thermo Fisher Scientific (9 mentions)
L glutamine, by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Sodium bicarbonate, by Corning (5 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Kanamycin, by Thermo Fisher Scientific (2 mentions)
Gentamycin, by Thermo Fisher Scientific (2 mentions)
0.22 μm filter, by GE Healthcare (2 mentions)
Tcm 199 medium, by Thermo Fisher Scientific (2 mentions)
Basic eagle s medium amino acids, by Thermo Fisher Scientific (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Fibronectin, by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Collagen 4, by Merck Group (1 mentions)
17 protocols using minimal essential medium nonessential amino acids
1
Primary Mouse RPE and Microglial Cell Isolation
2
Differentiation of iPSC-derived Brain Microvascular Endothelial Cells
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iPSC-derived brain microvascular endothelial-like cells (iBECs) were differentiated as previously described [33 (link), 44 (link)–46 , 48 (link)]. Briefly, iPSCs were seeded from a single cell suspension onto Matrigel coated cell culture flasks (Sarstedt) at a density of 1 × 104 cells/cm2 and expanded in StemFlex medium for 3 days with daily medium changes. Following iPSC expansion, the cells were differentiated in unconditioned medium [UM; DMEM/F-12 (Gibco), 20% Knockout serum replacement (Gibco), 1% minimal essential medium-nonessential amino acids (Gibco), 0.5% GlutaMAX (Gibco), and 0.07% beta-mercaptoethanol (Sigma)] for 6 days, changing medium daily. After 6 days, the medium was changed to basic EC medium [human endothelial cell serum-free media (Gibco) and 1% platelet-poor plasma derived serum (Fisher) or 1% B-27 supplement (Gibco)], supplemented with 20 ng/ml basic fibroblast growth factor (bFGF, ReproTech), and 10 μM all trans-retinoic acid (RA, Sigma), for 2 days. Finally, the differentiated iBECs were purified onto collagen IV (Sigma) and Fibronectin (Sigma) coated plates and transwells inserts (Corning; Greiner). Basic fibroblast growth factor and retinoic acid were removed one day after iBEC purification. Quality of differentiated iBECs was assessed via TEER measurements and immunofluorescence staining of characteristic BEC markers.
3
Culturing and Propagating BTV-1 in BSR Cells
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BSR cells (BHK-21 subclone, BHK21 cells (ATCC CCL10), HeLa cells (ATCC CCL-2), and sheep PT cells (ovine-derived kidney cells, ATCC CRL-1633) were maintained in Dulbecco modified Eagle medium (Sigma-Aldrich Co.). The medium was supplemented with 10% (vol/vol) fetal bovine serum (FBS; Invitrogen), 100 U of penicillin/ml and 100 μg of streptomycin/ml (Sigma-Aldrich Co.), and minimal essential medium nonessential amino acids (Gibco). BTV serotype 1 (BTV-1) stock was obtained by infecting BSR cells at a low MOI and harvested when a 100% cytopathic effect was evident. Virus stocks were stored at 4°C.
4
Culturing BxPC-3 Pancreatic Cancer Cells
5
Culturing Colon Cancer Cell Lines
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The human colon cancer cell lines HT-29 [12 (link)] and HCT 116 [13 (link)] were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin/streptomycin (Gibco-BRL, Carlsbad, CA), sodium pyruvate (Gibco-BRL), sodium bicarbonate (Cellgro, Manassas, VA), L-glutamine (Gibco-BRL), and minimal essential medium nonessential amino acids (Gibco-BRL). All cells were cultured at 37°C in a 5% CO2 incubator.
6
Stable GFP-Expressing Pancreatic Cancer Cells
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The human pancreatic cancer cell line BxPC-3 was stably transduced to express green fluorescent protein (GFP) as previously described [18 (link),19 (link)]. Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin/streptomycin (Gibco-BRL, Carlsbad, CA), sodium pyruvate (Gibco-BRL), sodium bicarbonate (Cellgro, Manassas, VA), l -glutamine (Gibco-BRL), and minimal essential medium nonessential amino acids (Gibco-BRL). All cells were cultured at 37°C in a 5% CO2 incubator.
7
Culturing Human Pancreatic Cancer Cells
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The human pancreatic cancer cell lines BxPC-3 [17 (link)] and Panc-1 [18 (link)] were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin/streptomycin (Gibco-BRL, Carlsbad, CA), sodium pyruvate (Gibco-BRL), sodium bicarbonate (Cellgro, Manassas, VA), L-glutamine (Gibco-BRL), and minimal essential medium nonessential amino acids (Gibco-BRL). All cells were cultured at 37° C in a 5% CO2 incubator.
8
Isolation and Culture of Mouse ESCs
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Embryonic stem cells (ESCs) were isolated from CIP2A and WT mouse blastocysts as described by Bryja and coworkers [53 ]. Briefly, time-mated females were killed at E3.5, and the blastocysts were flushed out of the uterine horn. Blastocysts were plated to dishes containing mitotically inactivated feeder cells (mouse embryonic fibroblasts, MEFs). Blastocysts were allowed to attach to MEFs and grow in ES medium containing knockout serum replacement (SR-ES medium). The content of the medium was: Knockout DMEM supplemented with 20% Knockout SR (Gibco), penicillin (100 U/ml)/streptomycin (100 g/ml) (Gibco), 2 mM L-glutamine (Gibco), 1 X minimal essential medium nonessential amino acids (Gibco), 100 μM-mercaptoethanol and recombinant mouse leukemia inhibitory factor (1,000 U/ml of ESGRO, Chemicon International, Temecula, CA). The blastocysts and ESCs derived from the inner cell mass of blastocysts were allowed to grow alternately in SR-ES medium and FCS-ES medium. In FCS-ES medium SR was replaced by 20% fetal calf serum (FCS). In the method, the cells were grown always after trypsinization in FCS-ES for one day to allow greater trophic support, whereas SR-ES medium supported selective propagation of ESCs between trypsinizations.
9
Differentiating gPSCs into Germ Layers
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To differentiate CI-gPSCs into three germ layers, previously described protocols (17 (link), 18 (link)) were applied to embryoid bodies derived from gPSCs. Embryoid bodies were attached to a gelatin-coated plate and cultured in MEF medium until beating cells were formed. MEF medium was Dulbecco’s modified Eagle’s medium (DMEM) low-glucose (Welgene, Gyeongsan, Korea) with the following supplements: 10% FBS (Gibco), 50 μM β-mercaptoethanol (Gibco), 1×penicillin–streptomycin (Welgene), and 1×minimal essential medium non-essential amino acids (Gibco).
10
Stable GFP Expression in BxPC-3 Pancreatic Cells
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