Beas‐2B human bronchial epithelial cells, two lung adenocarcinoma cell lines (H23 and HCC827), one squamous lung cancer cell line, H1703, and a large cell lung carcinoma cell line (H460) were obtained from ATCC® (catalog numbers: CRL‐9609, CRL‐5800, CRL‐2868, CRL‐5889, and HTB‐177). The lung adeno, squamous, and large cell carcinoma cell lines were maintained in an RPMI‐1640 medium according to the recommended protocol. Beas‐2B cells were cultured as described in our previous study.11 Briefly, the flasks, plates, and dishes for Beas‐2B culture were coated with 0.03 mg/ml PureCol (Sigma®), 0.01 mg/ml fibronectin (Sigma®), and 0.01 mg/ml bovine serum albumin (Sigma®) dissolved in BEBM for at least 2 h at 37°C, which was vacuum aspirated to dry. A BEGM‐completed growth medium containing the same concentrations of PureCol, fibronectin, and bovine serum albumin was used for culture of the cells at 37°C in 95% air and 5% CO2 atmosphere, which were incubated until reaching 80% confluence.
Purecol
Manufactured by Merck Group
Sourced in United States
PureCol is a type of laboratory equipment manufactured by Merck Group. It is designed for the purification and separation of biological samples. The core function of PureCol is to facilitate the extraction and isolation of specific target molecules or components from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Pneumacult ex plus medium, by STEMCELL (3 mentions)
Transwell insert, by Corning (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Fibronectin, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Primocin, by InvivoGen (1 mentions)
Pneumacult ali medium, by STEMCELL (1 mentions)
24 well ultra low attachment plate, by Corning (1 mentions)
Crystal violet solution, by Merck Group (1 mentions)
Transwell cell culture insert, by Corning (1 mentions)
8 protocols using purecol
1
Culturing Lung Cancer Cell Lines
2
Sprouting Angiogenesis Assay in 3D Spheroids
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HUVECs were cultured and siRNA transfection was performed as described above. Spheroids were formed from 1,000 cells/spheroid in MV2 cell culture medium (PromoCell) containing 5% FBS and 0.4% methylcellulose (2% stock solution) using a 96‐well round‐bottom plate (Costar, #3788). Spheroids were collected after 24 h and resuspended in a mixture of 1.5 mg/ml type I collagen (Advanced BioMatrix, PureCol), 0.4% methylcellulose (Sigma, #M7027). 2.5% FBS in polymerization buffer [Ham’s F12 medium‐Glutamax (Gibco), 12.5 mM NaOH, 1.25× F12 (Gibco), 25 mM HEPES, 0.146% NaHCO3. 1.25% Glutamax‐I (Gibco)]. Twenty‐five spheroids were seeded per well of a 24‐well plate on a thin 1.5 mg/ml collagen layer. After 1 h of gel polymerization at 37°C, 50 ng/ml VEGF‐A or control medium was applied. The gels were fixed after 24 h in 4% PFA for 1 h at room temperature or overnight at 4°C. Bright‐field images on an inverted microscope with 5× objective were acquired, and cumulative sprout length was measured by ImageJ.
3
Nasal Epithelial Cell Culture and Differentiation
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Nasal biopsies were performed with a 3-mm cytology brush (ConMed). Cells were dissociated from the brush by gentle agitation in PneumaCult-Ex Plus medium (STEMCELL Technologies, cat. no. 05040). The cells were seeded into a single well of a culture plate coated in collagen (PureCol, Sigma-Aldrich, cat. no. 5006–15MG). Once confluent, the cells were passaged and expanded further in a T25 flask. The cells were passaged a second time and seeded onto transwell inserts (Corning, cat. no. CLS3460) at a density of 24,000 cells per insert. Cells were cultured in PneumaCult-Ex Plus medium until confluent, at which time the media in the basal chamber was replaced with PneumaCult-ALI medium (STEMCELL Technologies, cat. no. 05001) and the apical surface exposed to provide an ALI. All media contained Primocin (InvivoGen, cat. no. ant-pm-05) and penicillin-streptomycin (Thermo Fisher Scientific, cat. no. 15070063) to prevent bacteria growth. Ciliation was observed 4 to 6 wk after transition to ALI. During differentiation, the basolateral media was refreshed every 2 to 3 d and the apical surfaces were washed with phosphate-buffered saline (PBS) to remove mucus.
4
Collagen Gel Contraction Assay
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Contraction assays to monitor matrix remodeling were as described (Goetz et al., 2011 (link), Orimo et al., 2005 (link)). Briefly, 1.5 × 105 MEFs were included in a collagen type I gel (PureCol, Sigma-Aldrich; 1.5mg/ml, 500 μL total volume) in an Ultra-Low Attachment 24-well plate (Corning; Corning, New York, United States). After gel polymerization, normal culture medium was added, and collagen gel borders were detached from the border of the plate. Gels were cultured at 37 °C, 5% CO2 for 48h. Gel contraction was monitored by quantifying the gel surface area on photographs with ImageJ. The fold-change with respect to the contraction observed in a control condition was calculated for each sample.
5
Nasal Epithelial Cell Culture Protocol
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Nasal brushing samples were taken from healthy participants turbinate using a 3-mm bronchial cytology brush under Health Research Authority study approval (REC ref: 20/SC/0208; IRAS: 282739). The nasal brushings were placed in PneumaCult-Ex Plus medium (STEMCELL Technologies, Cambridge, UK) and cells extracted from the brush by gentle agitation. The cells were seeded into a single well of a collagen (PureCol from Sigma Aldrich) coated plate and once confluent, they were passaged and expanded further in a T25 flask. The cells were passaged a second time and seeded onto Transwell inserts (6.5 mm diameter, 0.4 μm pore size, Corning) at a density of 24,000 cells per insert. Cells were cultured in PneumaCult-Ex Plus medium (STEMCELL Technologies, Cambridge, UK) until confluent before replacing with PneumaCult-ALI medium in the basal chamber and the apical surface exposed, giving an air liquid interface to stimulate cilia biogenesis.
6
Collagen-coated Transwell Assay for FLS Migration
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Transwell cell culture inserts (pore size 8 μm, Corning) were coated with MEM (Sigma) containing 40% collagen (PureCol, Sigma) at 37°C for 90 min. 2×104 FLS were seeded into the precoated insert in serum-free DMEM and introduced into a 24-well plate containing fully supplemented DMEM. After 36 h, the inserts were washed in PBS and subsequently incubated in crystal violet solution (Sigma) at RT in the dark for 20 min. Upon repeated washing and subsequent drying, the membranes were removed from the inserts and coated with Entellan solution (Millipore) on microscope slides. Pictures of whole membranes were obtained by binning in a 10x magnification with a Zeiss Axioobserver Z1. The ratio of the stained surfaces was calculated with the Biovoxxel Toolbox in the Fiji/ImageJ software.
7
Nasal Epithelial Cell Culture Protocol
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Nasal brushings were placed in PneumaCult-Ex Plus medium (STEMCELL Technologies, Cambridge, UK) and cells dissociated from the brush by gentle agitation. The cells were seeded into a single well of collagen (PureCol from Sigma-Aldrich) coated plate and once confluent, the cells were passaged and expanded further in a T25 flask. The cells were passaged a second time and seeded onto transwell inserts (6.5 mm diameter, 0.4 μm pore size, Corning) at a density of 24,000 cells per insert. Cells were cultured in PneumaCult-Ex Plus medium (STEMCELL Technologies, Cambridge, UK) until confluent, at which point the media was replaced with PneumaCult-ALI medium in the basal chamber and the apical surface exposed to provide an air-liquid interface (ALI). Ciliation was observed between 4–6 weeks post transition to ALI.
8
Culturing Tumor Cell Lines and LN Stromal Cells
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YUMM1.7 and YUMMER1.7 were provided by M.W. Bosenberg (Yale University) and cultured as previously described 59, 60 . MCA205 H12 sarcoma cells and MCA205.OVA were provided by A.E. Moran (Oregon Health & Science University; originally a gift of Suyu Shu) and cultured in DMEM supplemented with 10% FBS, 1% non-essential amino acids, 1mM sodium pyruvate, 10 mM HEPES, 1% penicillin/streptomycin. B16.F10 (ATCC CRL-6475), B16.F10.OVA (provided by M.A. Swartz at EPFL) and MC38 (provided by M.J. Gough at Providence Cancer Institute) tumor lines were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were maintained at 37°C with 5% CO 2 .
Primary lymph node (LN) stromal cells were cultured on tissue culture plates coated with 10 μg/ml PureCol (Sigma Aldrich) and 10 μg/ml fibronectin (Chemicon) in MEM (no nucleic acids) supplemented with 10% FBS and 1% penicillin and streptomycin. To induce CXCL12 gene expression 43 , LN stromal cells were cultured under hypoxic conditions (1% O 2 ) for 24 hours, and sorted for analysis with fluorescence-activated cell sorting (FACs).
Primary lymph node (LN) stromal cells were cultured on tissue culture plates coated with 10 μg/ml PureCol (Sigma Aldrich) and 10 μg/ml fibronectin (Chemicon) in MEM (no nucleic acids) supplemented with 10% FBS and 1% penicillin and streptomycin. To induce CXCL12 gene expression 43 , LN stromal cells were cultured under hypoxic conditions (1% O 2 ) for 24 hours, and sorted for analysis with fluorescence-activated cell sorting (FACs).
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