Primary human gingival epithelial cells (pHGEs) were purchased from CELLnTEC (catalog no. HGEPp; CELLnTEC, Research Triangle Park, NC) and maintained in the fully supplemented culture medium (catalog no. CnT-PR; CELLnTEC). Freshly prepared buffy coats were isolated from healthy donors by density gradient centrifugation as described previously (19 (link), 20 (link)). Briefly, mononuclear cells from 50 ml peripheral blood were purified with Ficoll Paque PLUS (catalog no. 17-1440-03; GE Healthcare, Pittsburgh, PA)–based density centrifugation. Purified cells were then incubated with magnetic-labeled CD14 beads (catalog no. 130-050-201; Miltenyi Biotec, San Diego, CA) to isolate monocytic cells according to manufacturer’s instruction. Purified monocytic cells from healthy donors were then plated at a density of 0.25 × 105 per well onto a 96-well plate and cultured in RPMI 1640 supplemented with penicillin–streptomycin and 10% FBS. For monocyte-derived macrophage differentiation, M-CSF (catalog no. 300-25-10UG; PeproTech, Rocky Hill, NJ) was added to the culture medium DMEM to achieve a final concentration of 100 ng/ml after monocytes were plated. Culture media supplemented with M-CSF was replenished every 72 h.
Cnt pr
Manufactured by CELLnTEC
Sourced in Switzerland
The CnT-PR is a compact and versatile laboratory instrument designed for cell culture applications. It features precise control of temperature, CO₂, and humidity levels to maintain optimal growth conditions for a wide range of cell types. The CnT-PR provides consistent and reliable performance, making it a valuable tool for researchers and cell culture professionals.
Automatically generated - may contain errors
Lab products found in correlation
Hgepp, by CELLnTEC (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
Hpeks, by CELLnTEC (2 mentions)
Magnetic labeled cd14 beads, by Miltenyi Biotec (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Cnt prime medium, by CELLnTEC (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Zyla 4.2 scmos camera, by Oxford Instruments (1 mentions)
Hyclone, by Thermo Fisher Scientific (1 mentions)
8 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Lipojet transfection reagent, by SignaGen (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Recombinant human tgf β1, by Thermo Fisher Scientific (1 mentions)
Y27632, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Fujifilm (1 mentions)
Mscgm cd, by Lonza (1 mentions)
Rneasy column, by Qiagen (1 mentions)
K sfm, by Thermo Fisher Scientific (1 mentions)
16 protocols using cnt pr
1
Isolation and Differentiation of Monocyte-Derived Macrophages
2
Limbal Biopsy Isolation and Culture
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Limbal biopsies were isolated and processed according to the protocol described by Haagdorens et al.85 (link). In brief, biopsies were taken from the superior and inferior keratolimbal region (Fig. 1 ) and washed for 30 minutes in CnT-prime medium (CnT-PR, CELLnTEC, Bern, Switzerland) at 4 °C. Explant cultures were initiated at the air-liquid interface, using CnT-PR medium, and cultured for 14 days at 37 °C and 5% CO2. From 2 days onward, cultures were submerged and the culture medium was changed every other day. This work flow results in a negligible fibroblast culture contamination, as previously shown86 (link). Prior to RNA extraction from the cultured limbal cells at day 14, limbal biopsies were removed using metal tweezers and cultures were rinsed with preheated PBS at 37 °C. Afterwards, cell lysis was performed according to RNeasy Micro kit guidelines (Qiagen, Hilden, Germany).
3
Limbal Stem Cell Cultivation on Novel Carriers
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Cadaveric donor eyes were collected from the cornea tissue bank of the Antwerp University Hospital. The donor age ranged from 49 to 90 years with an average of 74 years. All donor eyes were processed within 32 hrs postmortem. In brief, the eyes were enucleated, transferred in 0.9% NaCl, and stored at 4°C. The eyes were disinfected for 1 min in povidone iodine 0.5%, after which they were rinsed 4 times in PBS. Biopsies of ≤2 mm2 were taken from the superior and inferior keratolimbal regions and washed 6 × 10 min in CnT-PR (CELLnTEC) at 4°C. Biopsies were then placed epithelial side down on the tested carrier materials (Table 2 ) and cultivated for 14 days at 37°C, 5% CO2, and 95% humidity. For cultivations on HAM and RHC I, 1% hAB was added to culture medium. Culture medium was changed every other day. The first 3 days, cells were cultivated at an air-liquid interface to allow biopsy attachment. Onwards, volume of the medium was increased to submerge cultures. At day 14 (or earlier if confluent), cells were characterized through immunohistochemistry and reverse transcriptase PCR (RT-PCR) analyses.
4
Embryo Imaging Using Spinning Disk Confocal Microscopy
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Imaging was performed essentially as described previously37 (link). Briefly, embryos were dissected at E14.5 or E15.5 and immobilized on imaging chambers engineered on top of Lumox-Teflon imaging plates (Sarstedt). Whole embryos were immersed in growth medium (CnT-PR, CELLnTEC Advanced Cell Systems) supplemented with calcium-depleted 10% FBS (HyClone/Thermo Fisher), 20 U ml−1 penicillin-streptomycin (Thermo Fisher), 20 mM Hepes, 1.8 mM CaCl2 and 10 ng ml−1 human recombinant Epithelial growth factor (EGF) (Novus; NBP2-34952). Imaging was performed using an Andor Dragonfly 505 high-speed spinning disk confocal microscope (Oxford Instruments) equipped with 488-nm and 546-nm lasers, an Andor Zyla 4.2 sCMOS camera and an environmental chamber set at 37 °C, 5% CO2. Acquisition was carried out with ×25, ×40 dry or ×40 water-immersion objectives using the Fusion 2.0 software. After acquisition, the image series was 3D drift-corrected using manual registration on ImageJ together with the descriptor-based series registration ImageJ plug-in, before analysis. Image analyses are described in detail in the following. Optical cross-section live image sequences were generated using the oblique slicer tool on Imaris.
5
Comparative Cell Growth Analysis in HCT116
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Cell growth of HCT116-WT and the different clones were compared using an imaging method to determine percentage confluency with an IncuCyte ZOOM instrument (EssenBio, Welwyn Garden City, UK) using the Basic Analyzer software of the instrument. In brief, 50,000 cells were seeded in CnT-PR (CellnTec, Bern, Switzerland) supplemented with 5% FBS, in to 6well plates and confluency measured over 72 hours. Values are displayed as phase object confluence (percent) over time.
6
Regulation of DUOX1 by miR-125a-5p
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To determine the impact of miR-125a-5p on DUOX1, we overexpressed FAM-labeled locked nucleic conjugated miR-125a-5p mimics (Qiagen Inc) in primary human gingival epithelial cells pooled (HGEPp) (CELLnTEC Advanced Cell Systems AG, 3014 Bern, Switzerland) as DUOX1 protein expression was intense in the gingival epithelium. HGEPp cells were first cultured in CnT Prime medium (CnT-PR, CELLnTEC Advanced Cell Systems AG, Bern, Switzerland) in 8 well chamber slides (Thermo-Fisher Scientific Waltham, MA USA) at 37 °C in a humidified atmosphere with 5% CO2. At 95% confluency, CnT-Prime medium was replaced with CnT-Prime epithelial 2D differentiation medium (CnT-PR) and cultured for seven days to induce cellular differentiation. On day 8 of culture, cells were transfected with two different concentrations (20 and 30 nM) of FAM-LNA-miR-125a-5p or FAM-LNA-negative control mimic using the Lipojet transfection reagent (Signagen, DE). Cells were fixed with 2% paraformaldehyde at 96 h post-transfection and immunostained with DUOX1 and later with DAPI for nuclear localization.
7
Culturing and Characterizing Human Corneal Epithelial Cells
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Primary cultures of human CECs were established as previously described [25] (link). CECs were maintained with CnT-PR (CELLnTEC, Bern, Switzerland) with or without 20 ng/mL of recombinant human keratinocyte growth factor (KGF; Wako Pure Chemical Industries, Osaka, Japan) and 10 μM Y-27632 (Wako) for monolayer culture and DMEM:F12 (Life Technologies, Chesterfield, MO, USA) supplemented with 2% B-27 supplement, 20 ng/mL KGF, and 10 μM Y-27632 to reconstruct CEC sheets [26] (link). Recombinant human TGF-β1 used for induction of EMT into CECs was obtained from Peprotech (Rocky Hill, NJ, USA). Human AdMSCs were acquired from PromoCell (Heidelberg, Germany) and maintained with MSCGM-CD (Lonza) or Mesenchymal Stem Cell Growth Medium DXF (PromoCell). For the preparation of AdMSC-conditioned medium (CM), AdMSCs were cultured to 70–80% confluence in a T-75 flask. Subsequently, medium was changed to fresh medium. After 24–72 h of culture, the supernatant was collected, centrifuged at 300×g, and stored at −80 °C prior to use.
8
Gingival Epithelial Precursor Cell Infection
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Human gingival epithelial precursor cells obtained from pooled donors were purchased from CellnTec. The cells were cultured in epithelial medium (CellnTec CnT-PR) as submerged cultures on polystyrene lifts in 12-well plates. Cells were bathed with 800 µl media in the upper chamber of the lift and with 1 ml medium in the lower well. When the cells were confluent, viral stocks were added to the upper chamber and the infection was allowed to proceed for 24 h. Cells were monitored for eGFP expression and RNA was purified using RNeasy columns (Qiagen).
9
Cell Culture Protocols for iSLK, TREx-K-Rta BCBL-1, and Vero Cells
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iSLK cells54 (link) were maintained in DMEM medium containing 10% fetal bovine serum (FBS; complete DMEM), 50 μg/ml G418, and 100 μg/ml hygromycin B in the presence of 5% CO2. iSLK cells containing BAC16 WT or BAC16 mutants were cultured in complete DMEM containing 250 μg/ml G418, and 1000 μg/ml hygromycin and 1 μg/ml puromycin. TREx-(F3H3)-K-Rta BCBL-1 cells that expresses Flag × 3 and HA × 3 tags at the N-terminal region of K-Rta were generated and cultured in complete RPMI 1640 containing 50 μg/ml blasticidin and 100 μg/ml hygromycin B. Vero (ATCC CCL-81) and iVero (dox-inducible K-Rta expression) cells were cultured in the presence of 1 µg/ml puromycin. Human gingival epithelial cells (HGEP), obtained from pooled donors, were purchased from CellnTec (#HGEPp). The cells were cultured in CnT-PRime epithelial cell medium (CellnTec #CnT-PR).
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iSLK cells54 (link) were maintained in DMEM medium containing 10% fetal bovine serum (FBS; complete DMEM), 50 μg/ml G418, and 100 μg/ml hygromycin B in the presence of 5% CO2. iSLK cells containing BAC16 WT or BAC16 mutants were cultured in complete DMEM containing 250 μg/ml G418, and 1000 μg/ml hygromycin and 1 μg/ml puromycin. TREx-(F3H3)-K-Rta BCBL-1 cells that expresses Flag × 3 and HA × 3 tags at the N-terminal region of K-Rta were generated and cultured in complete RPMI 1640 containing 50 μg/ml blasticidin and 100 μg/ml hygromycin B. Vero (ATCC CCL-81) and iVero (dox-inducible K-Rta expression) cells were cultured in the presence of 1 µg/ml puromycin. Human gingival epithelial cells (HGEP), obtained from pooled donors, were purchased from CellnTec (#HGEPp). The cells were cultured in CnT-PRime epithelial cell medium (CellnTec #CnT-PR).
10
Isolation and Culture of Limbal Epithelial Cells
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Limbal biopsy segments were incubated at 4 °C overnight with 2 mg/ml of Dispase II (Roche) in KSFM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS. The limbal epithelium layer was then gently sloughed off from its stromal base under a dissecting microscope, incubated in TrypLE (Sigma) for 10 min and triturated with a pipette to generate an isolated cell suspension. These cells were seeded at a rate of 104 cells/cm2 in Cnt-Pr (Cell-N-Tec, Bern, Switzerland) complemented with bovine pituitary extract on 25 cm2 in T-flask coated with bovine collagen solution (PureColltm, Biomatrix, San Diego, CA).
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