For propagation, the human intestinal cell line HT29 MTX-E12 was cultured (at 37 °C, 5% CO2 −95% air) in RPMI containing 10% (v/v) FCS, 1% 5000 U (v/v) penicillin-streptomycin (Lonza). To form the in vitro colonic mucosal surface18 , 7.5 × 104 cells in 200 μl of RPMI containing 10% (v/v) FCS and penicillin-streptomycin were added to the apical side of Snapwell membranes (0.4 mm pores) with 12 mm diameter (Corning). When cells became confluent (4–6 days later) they were subjected to semi-wet interface culture with continuous rocking, with 2 ml media in the basolateral compartment and 50 μl of media in the apical compartment for 28 days. Basolateral media was refreshed every two days and for the first 6 days it was supplemented with 10 mM N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT, Sigma-Aldrich).
Snapwell membranes
Manufactured by Corning
Snapwell membranes are a type of lab equipment used for cell culture applications. They provide a permeable support for cell growth and migration. The membranes are made of a specialized material that allows for the exchange of nutrients, gases, and other substances necessary for cell viability and function.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Corning (2 mentions)
Penicillin streptomycin, by Lonza (2 mentions)
Transwell membrane, by Corning (2 mentions)
Recombinant epidermal growth factor and bovine pituitary extract, by Thermo Fisher Scientific (2 mentions)
Primaria plasticware, by BD (2 mentions)
Ht29 mtx e12, by American Type Culture Collection (1 mentions)
Rat tail collagen 1, by BD (1 mentions)
Mission shrna containing lentiviral particles, by Merck Group (1 mentions)
5 protocols using snapwell membranes
1
Colonic Mucosal Surface Modeling
2
Urothelial Cell Culture Protocol
3
In Vitro Mucosa Model Development
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The in vitro mucosal surface (Fig 1 ) was based on the colorectal adenocarcinoma HT29 MTX-E12 cell line (originated from HT29, American Type Culture Collection, USA, and was selected by growth adaptation to methotrexate and goblet cell morphology) and produced as previously described [14 (link)]. For expansion, the human intestinal cell line HT29 MTX-E12 was cultured (at 37°C, 5% CO2−95% air) in RPMI containing 10% (v/v) FCS, 1% 5000 U (v/v) penicillin-streptomycin (Lonza). To form the in vivo like in vitro mucosal surface [41 (link)], 7.5 x 104 cells in 200 μl of RPMI containing penicillin-streptomycin and 10% (v/v) FCS were added to the apical side of Snapwell membranes (0.4 mm pores) with 12 mm diameter (Corning). Four to 6 days later (when cells became confluent), the cells were subjected to semi-wet interface culture with continuous rocking, 50 μl of media in the apical compartment and 2 ml in the basolateral compartment for 28 days. Basolateral media was changed daily and supplemented with 10 mM N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT, Sigma-Aldrich) for the first 6 days and thereafter refreshed every two days.
4
Stable Cell Line Generation for Knockdown Studies
5
Urothelial Cell Culture Protocol
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Normal human urothelial (NHU) cells were isolated from human ureter and bladder biopsies and maintained as finite cell lines in vitro [20 (link),21 ]. Cultures were propagated on Primaria™ plasticware (BD Biosciences) in low calcium [0.09 mM] keratinocyte serum-free medium containing recombinant epidermal growth factor and bovine pituitary extract (Life Technologies), supplemented with 30 ng/ml cholera toxin (KSFMc) and used for experiments between passages 3-5. In these conditions, NHU cells proliferate as a monolayer that becomes contact-inhibited at confluence and can be propagated by serial, but finite sub-culture. Supplementing the medium to 2 mM Ca2+ (near physiological) results in stratification accompanied by the formation of adherens and tight junctions, but without urothelial cytodifferentiation [20 (link),21 ]. Urothelial cytodifferentiation with tight barrier formation was induced by subculturing the cells in KSFMc supplemented with 5% adult bovine serum and 2 mM Ca2+, as described [22 (link)]. In all cases, cultures were grown on 24 mm Transwell™ membranes (Corning) for mRNA and protein extractions and on 12 mm Snapwell™ membranes (Corning) for electrophysiological studies [23 (link)].
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