Dorsal skin was obtained from newborn mice and immersed in Dispase II solution, 2.5 U/ml Dispase II (Roche, Indianapolis, IN) with antibiotic-antimycotic solution CnT-ABM10 (CELLnTEC, Bern, Switzerland) in CnT-PR medium (CELLnTEC) for 16 h at 4°C. After being washed with PBS, the sheet of epidermis, separated from the dermis, was floated on a 500 μl drop of TrypLE select (Thermo Fisher Scientific) for 25 min at RT. After addition of 2 ml of medium to the drop, the sheet of epidermis was shaken gently on the drop to disperse the keratinocytes. The cell suspension was collected in a 15-ml tube containing fetal bovine serum (FBS). The cells were filtered through a 70-μm cell strainer to remove clumps and debris, centrifuged for 7 min at 200 × g, suspended in medium with 10% FBS and centrifuged again for 7 min at 1300 rpm. The cells were plated in CnT-PR medium with 10% FBS at 1.5 × 105 cells/cm2 and incubated at 37°C in a humidified 5% CO2 incubator. The medium was removed 16 h after plating and replaced with CnT-PR medium without FBS. The medium was changed every other day.
Cnt pr medium
Manufactured by CELLnTEC
Sourced in Switzerland
CnT-PR medium is a cell culture medium designed for the growth and maintenance of primary cells. It provides the necessary nutrients and growth factors to support the proliferation and viability of various primary cell types.
Automatically generated - may contain errors
Lab products found in correlation
Ker ct, by American Type Culture Collection (3 mentions)
Cnt pr 3d medium, by CELLnTEC (3 mentions)
Dispase 2, by Roche (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Tet system approved fbs, by Takara Bio (2 mentions)
Dmem f 12 medium, by Takara Bio (2 mentions)
Insulin transferrin selenium reagent, by Thermo Fisher Scientific (2 mentions)
Accutase, by Thermo Fisher Scientific (2 mentions)
Vk2 e6e7, by CELLnTEC (2 mentions)
Vk2 e6e7, by American Type Culture Collection (2 mentions)
Thincert, by Greiner (2 mentions)
Cnt pr 3d barrier, by CELLnTEC (2 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
Dispase, by Fujifilm (1 mentions)
12 well plate, by Greiner (1 mentions)
Smz645, by Nikon (1 mentions)
Epilife medium, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Il 13, by Thermo Fisher Scientific (1 mentions)
19 protocols using cnt pr medium
1
Isolation and Culture of Murine Keratinocytes
2
Human Keratinocyte Air-Liquid Culture
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Insert transwells (Merck, MCHT12H48) were seeded with 105 human foreskin keratinocytes (Ker-CT, ATCC CRL-4048) on the transwells in 300 μL CnT-PR medium (CellnTec) in a 12 well format. After 48 hours, cultures were switched to CnT-PR-3D medium (CELLnTEC, Bern 3014, Switzerland) for 24 hours and then cultured at the air–liquid interface for 17 days. From day 12 to 17 of the air–liquid interphase culture, the Th17 cytokines IL17A (30 ng/mL) and IL22 (30 ng/mL) were added [60 (link)]. Pharmacological treatments were applied from day 14 to 17 and consisted of 100 μM apocynin, 100 μM FK-866, 100 μM olaparib, 10 μM NP and 1 μM ATRA. Culture medium was refreshed every 2 days. At day 17, the tissues were harvested for gene expression analysis.
3
Isolation and Culture of Mouse Keratinocytes
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The keratinocytes used in this study were prepared as previously described (63 (link)), with minor modifications. Skin tissue was isolated from 1- or 2-day-old mice and incubated for 15–18 h at 4 ° C in CnT-PR medium (CELLnTEC; Bern, Switzerland) supplemented with antibiotic–antimycotic mixed solution and 5 mg/mL dispase (Wako Pure Chemical, Osaka, Japan). Subsequently, the epidermis was separated from the dermis, and keratinocytes were dissociated from the epidermis using Accutase. Postcollection, the keratinocytes were suspended in CnT-PR medium with antibiotic–antimycotic mixed solution and plated at a density of 3 × 105 cells/well in 12-well plates (Greiner Bio-One, GmbH, Germany) coated with collagen. The cells were cultured at 37 ° C in a 5% CO2 atmosphere. The culture medium was changed 16–20 h after plating (on day 3), and the cells were assayed on day 6.
4
Cell Culture Protocols for Various Cell Lines
5
Cell Culture Protocols for Various Cell Lines
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SCC13 cells (a gift from Paolo Dotto, MGH Cutaneous Biology department, USA) were grown in keratinocyte serum-free medium (K-SFM, Gibco) supplemented with EGF (epidermal growth factor) and bovine pituitary extract based on the manufacturer’s instruction. HSC2 cells (a gift from Cyril Benes, MGH Cancer Center, USA) were grown in DMEM/F-12 medium with 10% Tet system approved FBS (Clontech) and 1% penicillin (100 U/ml)/streptomycin (100 U/ml) (Gibco). Human primary keratinocytes were obtained from CellnTec and were grown in CnT-PR medium (CellnTec) supplemented with 1% penicillin (100 U/ml)/streptomycin (100 U/ml) (Gibco). Human tumor-associated fibroblasts (a gift from Salvador Aznar Benitah, IRB, Spain) were grown in DMEM medium supplemented with 10% FBS (Sigma), Insulin-Transferrin-Selenium reagent (Gibco), and 1% penicillin (100 U/ml)/streptomycin (100 U/ml) (Gibco). All cells were cultured and maintained at 37°C under 5% CO2. Human primary keratinocytes (Cat. #HPEKp, CellIn Tec), were passaged with accutase (Gibco) and were used within four passages. All the other cell lines were passaged by trypsinization.
6
Immortalized Vaginal Epithelial Cell Line Infection
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The human immortalized vaginal epithelial cell line (VK2/E6E7) was purchased from the American Type Culture Collection, and the Vero cell line was obtained from the Chinese Type Culture Collection. The VK2/E6E7 cells were cultured in Cn-TPR medium (CELLnTEC, Switzerland), and the Vero cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum. The cells were grown at 37°C in a 5% CO2 atmosphere.
HSV-2 strain 333 was propagated on a monolayer of Vero cells and stored at -80°C after harvest. We infected VK2/E6E7 cells with HSV-2 for different exposure times (6, 12, 18, 24,48 h), and 24 h post-infection was selected as the time point for the follow-up experiments based on the relevant results.
HSV-2 strain 333 was propagated on a monolayer of Vero cells and stored at -80°C after harvest. We infected VK2/E6E7 cells with HSV-2 for different exposure times (6, 12, 18, 24,48 h), and 24 h post-infection was selected as the time point for the follow-up experiments based on the relevant results.
7
Isolation and Cultivation of Human Epidermal Keratinocytes
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Normal human epidermal keratinocytes were isolated from neonatal skin. Frozen keratinocytes were thawed and cultivated at clonal density on a feeder layer of irradiated or mitomycin C–treated 3T3-J2 cells, at 37°C and 10% CO2 in a 3:1 mixture of the DMEM and Ham’s F12 medium supplemented with 10% FCS, 5 µg/ml insulin, 0.4 µg/ml hydrocortisone, 10−10 M cholera toxin, and 2 × 10−9 M triiodothyronine, as described previously (Rheinwald and Green, 1975 (link); Rochat et al., 1994 (link)). Human epidermal keratinocytes were also cultivated with CnT-PR medium (CELLnTEC), EpiLife medium containing supplement S7 (Life Technologies), and MCDB153 medium containing bovine pituitary extract (Hashimoto et al., 1994 (link)). Keratinocytes were used between passages 2 and 7. The medium was changed every 4 d. Cultures were fixed with 3.7% buffered formaldehyde and stained with 1% rhodamine B, and keratinocyte colonies were counted under a binocular microscope (SMZ645; Nikon). Clonal analysis was performed as described previously (Barrandon and Green, 1987b (link)). In brief, human keratinocytes were cultivated at clonal density on a feeder layer of irradiated or mitomycin C–treated 3T3-J2 cells. Well-isolated colonies were then individually trypsinized in a cloning ring and subcultured into a 10-cm cell culture dish so clonal types could be evaluated.
8
Airway Epithelial Cell Culture Protocol
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Insert transwells (Merck, Rahway, NJ, USA, MCHT12H48) were seeded with 200,000 human foreskin keratinocytes (Ker-CT, ATCC CRL-4048) on the transwells in 300 μL CnT-PR medium (CellnTec) in a 12-well format. After 48 h, the cultures were switched to CnT-PR-3D medium (CELLnTEC, Bern, Switzerland) for 24 h and then cultured in the air–liquid interface for 17 days. From day 12 to 17 of the air–liquid interphase culture, Th2 cytokines IL13 (10 ng/mL) and IL4 (10 ng/mL) (PeproTech, London, UK) were added [32 (link)]. Pharmacological treatments were applied from day 14 to 17 and consisted of 100 μM Olaparib, 100 μM FK-866 or their combination. The culture medium was refreshed every 2 days. On day 17, tissues were harvested for protein and gene expression analysis.
9
Tongue Epithelium Isolation and Immortalization
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Primary tongue epithelium from 3-week-old Rosa26-CreERT; Mob1aflox/flox; Mob1b−/− mice without TAM was obtained using a dermal keratinocyte isolation protocol (37 (link)). Briefly, resected tongue tissues were placed into ice-cold dispase digestion buffer [250 U of dispase (Godo Shusei) in PBS] and incubated overnight at 4°C. The epidermis was slowly separated from the dermis using forceps and floated on trypsin solution (Gibco) at room temperature for 40 min to create a primary tongue epithelial cell suspension. Tongue epithelial cells were cultured in CnT-PR medium (CELLnTEC) and passaged more than 40 times to generate the iMob1DKO tongue epithelial cell line. Loss of Mob1a/b in these cells in vitro was induced by treating them with TAM (0.5 μM; Toronto Research Chemicals) for 3 days.
10
Epidermal Barrier Formation in HEKn
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After 48‐h transfection, 3 × 105 HEKn cells were resuspended in CnT‐PR medium (CELLnTEC) and seeded onto Millicell PCF 12 mm inserts with 0.4 μm pore size (PIHP01250; Millipore). Twenty‐four hours later, medium was replaced inside and outside the insert with CnT‐PR‐3D medium (CELLnTEC). Twenty‐four hours later, air‐lifting was induced by removing the medium inside. Outside, medium was changed every 3 days during the following of 20 days of culture. Inserts were fixed in 10% buffered formalin solution, processed and paraffin‐embedded.
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