The human bronchial epithelial cell line Beas-2B and the NSCLC cell lines A549 and 95D were obtained from the Chinese Academy of Sciences’ typical culture preservation committee cell bank (Shanghai, China). Beas-2B cells were induced to undergo malignant transformation by exposure to NNK in our laboratory as previous report (designated as BEAS-2B-NNK) (30 (link)). Beas-2B and BEAS-2B-NNK cells were cultured in bronchial epithelial basal medium (BEBM, Clonetics/Lonza, Basel, Switzerland) supplemented with SingleQuots (Clonetics). A549 and 95D cells were cultured respectively in F-12 Kaighn’s Modification (HyClone, Logan, UT, USA) and RPMI-1640 (HyClone) media supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc., Chicago, IL, USA). All cells were cultured at 37°C in a humidified incubator in an atmosphere of 95% air and 5% CO2.
Singlequots
Manufactured by Cambrex
Sourced in United Kingdom, Switzerland, United States
The SingleQuots product is a lab equipment item designed for the accurate and convenient aliquoting of samples. It features a single-channel pipette for precise liquid handling and distribution. The core function of the SingleQuots is to enable the efficient division of samples into smaller, uniform portions for further analysis or processing.
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Lab products found in correlation
Tie 2 receptor, by BD (2 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Endothelial cell basal medium 2, by Lonza (1 mentions)
Bs 1 lectin binding, by Merck Group (1 mentions)
Kinase insert domain receptor kdr, by R&D Systems (1 mentions)
Pe labeled monoclonal mouse anti human antibodies recognizing cd31, by BD (1 mentions)
Kinase insert domain receptor, by R&D Systems (1 mentions)
H1299, by American Type Culture Collection (1 mentions)
Beas 2b, by American Type Culture Collection (1 mentions)
Beas 2b, by Merck Group (1 mentions)
Beas 2b, by Cambrex (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Hcasmc, by PromoCell (1 mentions)
9 protocols using singlequots
1
Beas-2B and NSCLC Cell Lines Cultivation
2
Isolation and Characterization of Human Late EPCs
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This study was approved by the Ethics Committee of First Affiliated Hospital of Jinan University. All donors provided written informed consent. Human late EPCs were purified from 20 healthy individuals and cultured following previously described methods.
31 (link) Briefly, peripheral blood mononuclear cells (PBMNCs) were maintained in endothelial cell basal medium‐2 (Lonza, Basel, Switzerland) supplemented with endothelial growth medium‐SingleQuots (Clonetics, San Diego, CA) in fibronectin‐coated 6‐well plates. After 4 days of culture, nonadherent cells were removed. The adherent cells were cultured for 28 days, with the medium being refreshed every 7 days. After 28 days of culture, human late EPCs were characterized by a double‐positivity for BS‐1 lectin binding (0.01 mg/mL; Sigma‐Aldrich, St. Louis, MO) and Dil‐acetylated low‐density lipoprotein uptake (0.02 mg/mL; Invitrogen, Carlsbad, CA). Cultured EPCs were incubated with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindo‐carbocyanine‐acetylated low‐density lipoprotein for 2 hours in a cell incubator. Subsequently, cells were washed and fixed with 4% paraformaldehyde for 15 minutes and incubated with FITC‐BS‐1 lectin for 1 hour. Plates of cells were again washed and incubated with DAPI nuclear counterstain. Double‐positive cells were observed with a fluorescent microscope (×200 magnification; Olympus, Tokyo, Japan).
31 (link) Briefly, peripheral blood mononuclear cells (PBMNCs) were maintained in endothelial cell basal medium‐2 (Lonza, Basel, Switzerland) supplemented with endothelial growth medium‐SingleQuots (Clonetics, San Diego, CA) in fibronectin‐coated 6‐well plates. After 4 days of culture, nonadherent cells were removed. The adherent cells were cultured for 28 days, with the medium being refreshed every 7 days. After 28 days of culture, human late EPCs were characterized by a double‐positivity for BS‐1 lectin binding (0.01 mg/mL; Sigma‐Aldrich, St. Louis, MO) and Dil‐acetylated low‐density lipoprotein uptake (0.02 mg/mL; Invitrogen, Carlsbad, CA). Cultured EPCs were incubated with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindo‐carbocyanine‐acetylated low‐density lipoprotein for 2 hours in a cell incubator. Subsequently, cells were washed and fixed with 4% paraformaldehyde for 15 minutes and incubated with FITC‐BS‐1 lectin for 1 hour. Plates of cells were again washed and incubated with DAPI nuclear counterstain. Double‐positive cells were observed with a fluorescent microscope (×200 magnification; Olympus, Tokyo, Japan).
3
Isolation and Characterization of Late Endothelial Progenitor Cells
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PBMNCs were isolated by Ficoll density gradient centrifugation from healthy subjects and CAD patients as described previously, and late EPCs were cultured and characterized by following the protocol described [15 (link), 16 (link)]. In short, PBMCs were cultured on fibronectin-coated 6-well plates in EBM-2 (contents: ascorbic acid 0.5 ml, rhFGF-B 2.0 ml, heparin 0.5 ml, GA-1000 0.5 ml, rhEGF 0.5 ml, hydrocortisone 0.2 ml, VEGF 0.5 ml, R3-IGF-1 0.5 ml) supplemented with endothelial growth medium-SingleQuots (Clonetics, San Diego, CA, USA). For all assays, late EPCs were used at passage 3 (approximately 28 days) after being removed by complete washing with culture medium. After 28 days, endothelial markers of cultured EPCs were examined by flow cytometry analysis using CD31 (BD Biosciences, San Jose, CA, USA), Tie-2 receptor (BD Biosciences), and kinase-insert domain receptor (KDR) (R&D Systems, Inc., Minneapolis, MN, USA). EPCs isolated from healthy and CAD patients separately were both cultured at 37 °C and 5% CO2 in EBM-2, and the medium was changed every 48 h.
4
Endothelial Cell Culture Protocol
5
Late EPC Isolation and Characterization
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Late EPCs were cultured and characterized as in our previous study [20 (link)]. Briefly, PBMCs from healthy subjects were cultured on fibronectin-coated 6-well plates in EBM-2 supplemented with endothelial growth medium-SingleQuots (Clonetics, San Diego, CA, USA). After a 4-day culture, non-adherent cells were removed by thoroughly washing with culture medium. Medium was changed daily for 7 days, and then every other day until the first passage. For all assays, LEPCs were used at passages 3 (about 28 days). After 28 days of culture, marker proteins of cultured EPCs were examined by flow cytometry analysis using phycoerythrin (PE)-labeled monoclonal mouse anti-human antibodies recognizing CD31 (BD Pharmingen), Tie-2 receptor (BD Pharmingen) and kinase-insert domain receptor (KDR) (R&D system). Overall, (92.25 ± 4.8)% of the cells were positive for CD31, (80.16 ± 6.5)% for KDR, and (88.36 ± 5.6)% for Tie-2 receptor. Based on the isolation and cultivation protocol, the adherent mononuclear cells were identified as late EPCs.
6
NNK-Induced Lung Cancer Cell Transformation
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The lung cancer cell lines 95D, A549, H1299 and the human bronchial epithelial cell line BEAS-2B were obtained from American Type Culture Collection (Manassas, VA, USA).
BEAS-2B cells were treated with NNK (Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 100 mg/L. After 24 h of exposure, the culture medium was discarded, and the cells were washed twice with PBS (PBS) and grown in fresh complete culture medium. When the cells were 80% confluent, the cells were passaged. When the cells were 70% confluent, the above NNK exposure steps were repeated and the cells were passaged to the sixth generation. The NNK-induced malignant transformation of the BEAS-2B cells was detected using the soft-agar clone formation rates. And these cells were designated as 2B-NNK cells.
BEAS-2B and 2B-NNK cells were grown in bronchial epithelial basal medium (BEBM; Clonetics, Basel, Switzerland) supplemented with SingleQuots (Clonetics). 95D and H1299 cells were grown in RPMI-1640 medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA). A549 cells were grown in F-12K medium (HyClone) supplemented with 10% FBS. All cells were incubated at 37°C in a humidified incubator containing 5% CO2.
BEAS-2B cells were treated with NNK (Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 100 mg/L. After 24 h of exposure, the culture medium was discarded, and the cells were washed twice with PBS (PBS) and grown in fresh complete culture medium. When the cells were 80% confluent, the cells were passaged. When the cells were 70% confluent, the above NNK exposure steps were repeated and the cells were passaged to the sixth generation. The NNK-induced malignant transformation of the BEAS-2B cells was detected using the soft-agar clone formation rates. And these cells were designated as 2B-NNK cells.
BEAS-2B and 2B-NNK cells were grown in bronchial epithelial basal medium (BEBM; Clonetics, Basel, Switzerland) supplemented with SingleQuots (Clonetics). 95D and H1299 cells were grown in RPMI-1640 medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA). A549 cells were grown in F-12K medium (HyClone) supplemented with 10% FBS. All cells were incubated at 37°C in a humidified incubator containing 5% CO2.
7
Culturing Human Endothelial and Smooth Muscle Cells
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Human umbilical vein endothelial cells (HUVECs) were purchased from TCS CellWorks (Bucks, United Kingdom) and cultured in endothelial basal medium (Cambrex BioScience Ltd, Nottingham, United Kingdom) supplemented with gentamicin-ampicillin, epidermal growth factor, bovine brain extract (Singlequots; Cambrex), and 10% fetal bovine serum (Life Technologies, Paisley, United Kingdom). HUVECs used in experiments were no more than passage 6. Human coronary artery smooth muscle cells were purchased from PromoCell (Heidelberg, Germany).
8
Cell Culture Protocols for Various Cell Lines
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Human umbilical vein endothelial cells
(HUVECs) were obtained from TCS Cell Works (Buckingham, UK) and were
cultured on tissue culture-coated flaks in endothelial basal medium
(EBM; Cambrex BioScience Ltd., Nottingham, UK) supplemented with gentamycin–ampicillin,
epidermal growth factor, and bovine brain extract (Singlequots; Cambrex)
and 10% fetal bovine serum (FBS) (complete EBM). For experimental
purposes, fully confluent HUVECs at passages 1–3 were preincubated
overnight with 1% FBS in EBM prior to addition of factors and other
treatments. DU145/cells.Ad.NRP1 cells: DU145 prostate cancer cells
were from ATCC and infected with an adenovirus expressing WT NRP1
as previously described27 (link) (cells were grown
in 10% FCS in DMEM). A375P melanoma cells were from ATCC. Cells were
grown in 10% FCS in DMEM.
Human dermal fibroblasts (HDF) were
obtained from TCS cells Works Buckingham, UK) and were cultured in
Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented
with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco).
For experimental purposes, fully confluent HDF at passages 1–3
were preincubated overnight with serum-free DMEM prior to other procedures.
GL261 cells were purchased from ATCC and cultured as described.44 (link),45 (link)
(HUVECs) were obtained from TCS Cell Works (Buckingham, UK) and were
cultured on tissue culture-coated flaks in endothelial basal medium
(EBM; Cambrex BioScience Ltd., Nottingham, UK) supplemented with gentamycin–ampicillin,
epidermal growth factor, and bovine brain extract (Singlequots; Cambrex)
and 10% fetal bovine serum (FBS) (complete EBM). For experimental
purposes, fully confluent HUVECs at passages 1–3 were preincubated
overnight with 1% FBS in EBM prior to addition of factors and other
treatments. DU145/cells.Ad.NRP1 cells: DU145 prostate cancer cells
were from ATCC and infected with an adenovirus expressing WT NRP1
as previously described27 (link) (cells were grown
in 10% FCS in DMEM). A375P melanoma cells were from ATCC. Cells were
grown in 10% FCS in DMEM.
Human dermal fibroblasts (HDF) were
obtained from TCS cells Works Buckingham, UK) and were cultured in
Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented
with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco).
For experimental purposes, fully confluent HDF at passages 1–3
were preincubated overnight with serum-free DMEM prior to other procedures.
GL261 cells were purchased from ATCC and cultured as described.44 (link),45 (link)
9
Culturing Human Umbilical Vein Endothelial Cells
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