BEAS-2B cells were purchased from American Type Culture Collection (ATCC® CRL-9609TM, In Vitro Technologies, Melbourne, Victoria, Australia) were cultured in BEGMTM bronchial epithelial cell growth medium BulletKitTM (CC3170, Lonza, Mount Waverley, Victoria, Australia) containing BEGMTM basal medium (CC-3171, Lonza, Mount Waverley, Victoria, Australia) and BEGMTM SingleQuotsTM supplements (CC-4175, Lonza, Mount Waverley, Victoria, Australia) supplemented with 1% penicillin/streptomycin (P4333, Sigma, North Ryde BC, New South Wales, Australia) and 1% amphotericin B (A2942, Sigma, North Ryde BC, New South Wales, Australia). Human small airway epithelial cells (SAECs) (CC-2547, purchased from Lonza, Mount Waverley, Victoria, Australia) were cultured in SAGMTM small airway epithelial cell growth medium BulletKit (CC-3118, Lonza, Mount Waverley, Victoria, Australia) containing SABMTM basal medium (CC-3119, Lonza, Mount Waverley, Victoria, Australia) and SAGM SingleQuotsTM supplements (CC-4124, Lonza, Mount Waverley, Victoria, Australia) supplemented with 1% penicillin/streptomycin (P4333, Sigma, North Ryde BC, New South Wales, Australia) and 1% amphotericin B (A2942, Sigma, North Ryde BC, Australia), grown in flasks coated in bovine collagen I (A10644-01, Life Technologies, Scoresby, Victoria, Australia). Cells were maintained in a humidified incubator (37 °C, 5% CO2).
A2942
Manufactured by Merck Group
Sourced in United States, Australia
A2942 is a laboratory equipment product offered by Merck Group. It serves as a core function in various research and analytical applications. The specific details and intended use of this product are not provided in order to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
P4333, by Merck Group (4 mentions)
F7524, by Merck Group (3 mentions)
R8758, by Merck Group (2 mentions)
S0615, by Merck Group (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
G7513, by Thermo Fisher Scientific (2 mentions)
D6419, by Merck Group (2 mentions)
Fungizone, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
G7513, by Merck Group (1 mentions)
P4458, by Merck Group (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
D6429, by Merck Group (1 mentions)
9 protocols using a2942
1
Culturing Human Airway Epithelial Cells
2
Murine Melanoma and Lung Carcinoma Cell Culturing
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Murine melanoma cells B16-F10 (ATCC, #CRL6475) and Lewis lung carcinoma (LCC1) cell line (ATCC, #CRL1642) were kept in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (41966, GIBCO, Billings, MT, USA), buffered at pH 7.2–7.4, and supplemented with 10% fetal bovine serum (GIBCO, USA), penicillin–streptomycin solution (104 U/mL and 10 mg/mL, respectively) (P4333, Sigma–Aldrich, St. Louis, MO, USA), and amphotericin B solution (250 mg/mL) (A2942, Sigma-Aldrich). Cell expansion was achieved by removing the whole medium, detaching the cells by rinsing the culture with 0.25% (w/v) Trypsin–EDTA (1× solution (25200, GIBCO, USA), and plating aliquots of the cell suspension in flasks incubated at 37 °C and 5% CO2.
3
Culturing A549 Lung Cancer Cells
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Commercially accessible epithelial lung carcinoma cells from the ATCC were used in this investigation, A549 (ATCC®, CCL185TM). The media used for maintaining the lung cancer cells and isolated CSCs comprised the accompanying: Rosewell Park Memorial Institute 1640 medium (RPMI-1640) (SIGMA, R8758) enhanced with 10% fetal bovine serum (Biochrom, S0615) and 0.5% penicillin/streptomycin (SIGMA, P4333) and 0.5% amphotericin B (SIGMA, A2942). All cultured cells were maintained and incubated at 37°C in 5% CO2 and 85% humidity.
4
Isolation and Culture of Human Lung Fibroblasts
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For in vitro experiments using human lung fibroblasts, cells from four to six donors were used per group. Informed consent was obtained from all donors and work was approved by the East Midlands Nottingham 1 Research Ethics Committee (reference 08/H0407/1). Cells were used at passage 5-6 for all in vitro experiments.
HLFs were isolated from donated post-mortem or surgical lung biopsy samples, from male and female donors with and without pulmonary fibrosis. For non-fibrotic fibroblasts, cells were isolated from regions of lung distant from the area of primary diagnosis. Tissue was cut into 1 mm×1 mm pieces and placed 10 mm apart in a 10 cm cell culture dish. Tissue was cultured in DMEM supplemented with 10% foetal calf serum (FCS, Fisher Scientific, 11573397), L-glutamine (4 mM, G7513, Sigma-Aldrich), penicillin (200 units/ml) and streptomycin (0.2 mg/ml) (P4458, Sigma-Aldrich) and amphotericin B (2.5 µg/ml, A2942, Sigma-Aldrich). Fibroblast outgrowth could be seen after 6-8 days. Tissue was removed from the cell culture dish if it became detached, or when cells had reached 80% confluency and were ready for passage. Cells were maintained in a humidified incubator at 37°C, 5% CO2/95% air, in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich), supplemented with 10% FCS, L-glutamine (4 mM), penicillin (100 units/ml) and streptomycin (0.1 mg/ml).
HLFs were isolated from donated post-mortem or surgical lung biopsy samples, from male and female donors with and without pulmonary fibrosis. For non-fibrotic fibroblasts, cells were isolated from regions of lung distant from the area of primary diagnosis. Tissue was cut into 1 mm×1 mm pieces and placed 10 mm apart in a 10 cm cell culture dish. Tissue was cultured in DMEM supplemented with 10% foetal calf serum (FCS, Fisher Scientific, 11573397), L-glutamine (4 mM, G7513, Sigma-Aldrich), penicillin (200 units/ml) and streptomycin (0.2 mg/ml) (P4458, Sigma-Aldrich) and amphotericin B (2.5 µg/ml, A2942, Sigma-Aldrich). Fibroblast outgrowth could be seen after 6-8 days. Tissue was removed from the cell culture dish if it became detached, or when cells had reached 80% confluency and were ready for passage. Cells were maintained in a humidified incubator at 37°C, 5% CO2/95% air, in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich), supplemented with 10% FCS, L-glutamine (4 mM), penicillin (100 units/ml) and streptomycin (0.1 mg/ml).
5
Cell Culture Protocols for Adherent and Suspension Cell Lines
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The adherent cell lines Hek293 (Human embryonic kidney, ATTC® CRL-1573, ATCC, Manassas, VA, USA) and HeLa S3 (Cervical cancer, ATTC® CCL-2.2) were cultured in DMEM high glucose (D6429, Sigma-Aldrich, St. Louis, MO, USA). The suspension cell lines NB4 (Acute promyelocytic leukemia, kindly gifted by Professor Stein Døskeland, University of Bergen, Bergen, Norway [68 (link)]) and MC/CAR (Myeloma, ATTC® CRL-8083) were cultured in RPMI-1640 (R8758, Sigma-Aldrich) and IMDM (21980-032, Thermo Fisher Scientific, Waltham, MA, USA) media, respectively. All culture media were supplemented with 2 mM glutamine (K0283, VWR, Radnor, PA, USA), 100 µg/mL gentamicin (G1272, Sigma-Aldrich), 2.5 µg/mL amphotericin (A2942, Sigma-Aldrich), and fetal bovine serum (F7524, Sigma-Aldrich), 10% in DMEM high glucose and RPMI-1640, and 20% in IMDM. All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2.
6
Cell Culture Conditions for Experimental Purposes
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Cells were cultured in Rosewell Park Memorial Institute 1640 medium (RPMI-1640) (Sigma-Aldrich, R8758) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, S0615) and 0.5% penicillin/streptomycin (Sigma-Aldrich, P4333) and 0.5% amphotericin B (Sigma-Aldrich, A2942). Cell lines enriched for the respective markers were maintained at a passage between 4 and 8 for all experimental purposes. All cultures were maintained and incubated at 37 °C in 5% CO2 and 85% humidity.
7
Cultivation of Cell Lines for Research
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All cell lines were obtained from the American Type Culture Collection (ATCC) and cultivated in a humidified incubator at 37 °C and 5% CO2. The human keratinocyte cell line HaCaT was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, D6419) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, F7524), 2 mM glutamine (Sigma-Aldrich, G7513), 0.1 mg/ml gentamicin (Gibco, 15710049), and 1.25 µg/ml fungizone (Sigma-Aldrich, A2942). For the lung carcinoma cell line A549 we used DMEM supplemented with 10% FBS and 2 mM glutamine, while the colorectal carcinoma cell line LS411N and the colorectal adenocarcinoma cell line DLD1 were cultivated in RPMI 1640 medium (Gibco, A1049101) supplemented with 10% FBS.
8
Cell Culture Conditions for Diverse Cell Lines
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All cell lines were obtained from the American Type Culture Collection (ATCC) and cultivated in a humidified incubator at 37°C and 5% CO2. The human keratinocyte cell line HaCaT was cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, D6419) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, F7524), 2 mM glutamine (Sigma-Aldrich, G7513), 0.1 mg/ml gentamicin (Gibco, 15710049), and 1.25 µg/ml fungizone (Sigma-Aldrich, A2942). For the lung carcinoma cell line A549 we used DMEM supplemented with 10% FBS and 2 mM glutamine, while the colorectal carcinoma cell line LS411N and the colorectal adenocarcinoma cell line DLD1 were cultivated in RPMI 1640 medium (Gibco, A1049101) supplemented with 10% FBS.
9
Isolation of Human Umbilical Vein Endothelial Cells
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Human umbilical cords were obtained following elective Caesarean sections. Saline was used to flush blood out of the umbilical vein, before one end was clamped.
Collagenase type 1A (1 mg/mL; C5894, Sigma) in Medium 199 (M199; 31150, ThermoFisher) at 37 °C, was syringed into the vein until the cord became taut and then the end of the cord was also clamped. After 15 minutes, one clamp was released allowing the vascular endothelial cell suspension to be collected into a Falcon tube. Copious growth medium (M199 containing 10 % (v/v) foetal bovine serum (10500-064, ThermoFisher), human epidermal growth factor (1 ng/mL; 13453029, ThermoFisher), hydrocortisone (1 µg/mL; H0888, Sigma), gentamycin (35 µg/mL; G1272, Sigma) and amphotericin (0.5 µg/mL; A2942, Sigma)) was added to terminate the enzymatic digestion. Cells were centrifuged (300 g, 5 minutes, 4 °C), resuspended in growth medium, and plated in 96-well plates for leukocyte attachment assays or 6-well plates for protein assays. Plates were pre-coated with 1 % (w/v) bovine skin gelatin (G9391, Sigma) in 1× PBS. HUVECs were given 2 hours to adhere, before the medium was aspirated and replaced. HUVECs reached confluency within 4-7 days, were never passaged, and were used for experiments within 7 days.
Collagenase type 1A (1 mg/mL; C5894, Sigma) in Medium 199 (M199; 31150, ThermoFisher) at 37 °C, was syringed into the vein until the cord became taut and then the end of the cord was also clamped. After 15 minutes, one clamp was released allowing the vascular endothelial cell suspension to be collected into a Falcon tube. Copious growth medium (M199 containing 10 % (v/v) foetal bovine serum (10500-064, ThermoFisher), human epidermal growth factor (1 ng/mL; 13453029, ThermoFisher), hydrocortisone (1 µg/mL; H0888, Sigma), gentamycin (35 µg/mL; G1272, Sigma) and amphotericin (0.5 µg/mL; A2942, Sigma)) was added to terminate the enzymatic digestion. Cells were centrifuged (300 g, 5 minutes, 4 °C), resuspended in growth medium, and plated in 96-well plates for leukocyte attachment assays or 6-well plates for protein assays. Plates were pre-coated with 1 % (w/v) bovine skin gelatin (G9391, Sigma) in 1× PBS. HUVECs were given 2 hours to adhere, before the medium was aspirated and replaced. HUVECs reached confluency within 4-7 days, were never passaged, and were used for experiments within 7 days.
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