siRNA probes targeting sfrp2 in human mesenchymal cells and frizzled-5 and −6 in AEC2 cells were obtained from Thermo Fisher (Supplemental Table 4 ). In brief, 20,000 hAEC2 were incubated for 4 hrs in Opti-DMEM (cat. no. 31985062, Fisher Scientific) with 1pmole of siRNA probes using Lipofectamine RNAiMAX (cat. no. 13778, Invitrogen). Cells were then plated on growth factor-reduced Matrigel (cat. no. CB-40230A, Thermo Fisher) cultured in small airway basal medium (SABM, cat. no.CC-3118, Lonza) with BPE low protein, insulin, transferrin, retinoic acid, epinephrin, triiodothyronine, and epidermal growth factor as per the SAGM bullet kit (cat. no. CC-3118, Lonza) supplemented with KGF (cat. no. 251KG01050, R&D, 100 ng ml−1), 5% FBS and 1% Pen/Strep for 48 hrs.
Sagm bulletkit
Manufactured by Lonza
Sourced in Switzerland
The SAGM BulletKit is a laboratory equipment product offered by Lonza. It is designed to provide a complete system for the culture and maintenance of human airway epithelial cells. The kit includes the necessary components and reagents required for cell growth and differentiation, without further interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Rock inhibitor, by Merck Group (2 mentions)
Matrigel, by Merck Group (2 mentions)
Cholera toxin, by Merck Group (2 mentions)
Growth factor reduced matrigel, by Corning (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Egm 2mv, by Lonza (1 mentions)
Caseinolytic units, by Corning (1 mentions)
Dispase, by Corning (1 mentions)
Pirfenidone, by R&D Systems (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Ebm 2 plus singlequots of growth supplements, by Lonza (1 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (1 mentions)
Egm 2 bulletkit, by Lonza (1 mentions)
Hsaec, by Lonza (1 mentions)
Begm bullet kit, by Lonza (1 mentions)
Lenti x 293t cell line, by Takara Bio (1 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Rpmi 1640, by Nacalai Tesque (1 mentions)
11 protocols using sagm bulletkit
1
Knockdown of sfrp2, frizzled-5 and -6 in hAEC2
2
Bronchial Epithelium Co-culture Assay
3
Human Airway and Endothelial Cell Culture
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Human small airway epithelial cell (HSAEC) (Lonza, CC-2547, Basel, Switzerland) were grown in SAGM™ BulletKit™ (Lonza, cat. CC-3118, Basel, Switzerland) in 5% CO2/95% air at 37 °C. Human lung microvascular endothelial cells (HLMVEC) (PromoCell cat. C12281, Heidelberg, Germany) were grown in EGM-2MV (Lonza, cat. CC-3202, Basel, Switzerland) in 5% CO2/95% air at 37 °C. Under these growth conditions the media pyruvate and glucose concentrations are 0.6 mM and 6 mM, respectively, for HSAEC; and 1.2 and 5.5 mM, respectively, for HLMVEC.
4
Isolation of Alveolar Epithelial Cells
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Primary cells were harvested from conditional Nedd4-2−/− mice and littermate controls after the indicated period of doxycycline induction. Dispase (5000 caseinolytic units, Corning) was instilled through the trachea and incubated for 40 min at room temperature. After mincing the lung, the suspension was filtered sequentially through 100, 40, and 10-µm nylon meshes and centrifuged at 130 × g for 8 min. The pellet was resuspended in DMEM/0.025 M HEPES. To separate macrophages, lymphocytes and endothelial cells from epithelial cells, the cell suspension was incubated with biotinylated antibodies against Cd45, Cd16/32, and Cd31 (BD Pharmingen). Obtained Cd45, Cd16/32 and Cd31 negative cells were plated on collagen-coated 24-well plates (Corning BioCoat) at 500,000 cells per well, cultured with AEC medium (SAGM Bullet Kit, Lonza) in a humidified atmosphere of 5% CO2 at 37 °C and medium was changed every second day. On day 7, medium was replaced with growth factor-free medium, with or without pirfenidone (1 mg/ml; TCI) for 3 h, followed by stimulation with 1 ng/ml TGFβ (R&D Systems). Subsequently, cells were subjected to RNA or protein isolation.
5
Cultivation of Human Cell Lines
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Human umbilical vein endothelial cells (HUVEC) (Lonza Walkersville, Inc.), human small airway epithelial cells (SAEC) (Lonza Walkersville, Inc., Walkersville, MD), and skeletal muscle cells (SkMC) (Lonza Walkersville, Inc.) were harvested from cryopreserved batches from our laboratory, plated and grown confluent with the following mediums: EGM-2 Bulletkit, EBM-2 plus SingleQuots® of growth supplements (CC-3162) (Lonza Walkersville, Inc.), SAGM Bulletkit, SABM plus SingleQuots® of growth supplements (CC-3118) (Lonza Walkersville, Inc.), and skeletal muscle cell supplemented growth medium (Cat No. 151–500) (Cell Applications, Inc.).
6
Establishment of Immortalized Human Lung Epithelial Cells
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The establishment of HSAEC_4T53RD cells has been described previously [10 (link)]. Briefly, normal HSAECs, purchased from Lonza (Walkersville, MD, USA), were malignantly transformed by the introduction of Cdk4, hTERT, a dominant negative p53 mutant, K-rasV12, and cyclin D1, using retroviral vectors. The cells were cultured on collagen-coated dishes in serum-free SAGM medium supplemented with growth factors supplied by the manufacturer (SAGM Bullet Kit; Lonza), and were maintained at 37°C in a low-oxygen environment (3% O2 and 5% CO2) in a humidified incubator. TIG-3 human lung fibroblasts were obtained from the Japanese Collection of Research Bioresources Cell Bank and were cultured in DMEM supplemented with 10% FBS at 37°C in a low-oxygen environment (3% O2 and 5% CO2) in a humidified incubator.
7
Bronchial Epithelium Co-culture Assay
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GFP+ bronchial epithelium were FACS sorted from Scgb1a1cre:R26RmTmG lungs and co-cultured with lung mesenchyme isolated from UBCcreERT2:R26RSmoM2 animals (5×103 epithelial cells:5×104 mesenchymal cells/well) in a modified MTEC media diluted 1:1 in growth factor reduced Matrigel (Corning). Modified MTEC culture media is comprised of small airway basal media (SABM) (Lonza) with selected components from SAGM bullet kit (Lonza) including insulin, transferrin, bovine pituitary extract, retinoic acid, and gentamicin/amphotericin B. Additional components include 25 ng/mL mEGF (Sigma), 0.1 ug/mL cholera toxin (Sigma), and 5% FBS (Life Technologies). Cell suspension-Matrigel mixture is placed in a transwell and incubated in growth media with 10 uM ROCK inhibitor (Sigma) in a 24 well plate with vehicle or 1ug/ml 4-OH-tamoxifen for 48 hours, after which the media was replenished every 48 hours (lacking tamoxifen). Colonies are assayed after 14 days. Each experimental condition is performed in quadruplicates and counted blinded to the experimental condition. Colony forming efficiency = (number of GFP+ colonies/number GFP+ epithelial cells cultured per well) × 100. Areas of individual colonies are assayed on ImageJ and over 140 colonies are randomly sized per experimental condition.
8
Culturing and Differentiating Bronchial Epithelial Cells
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Human BEAS-2B bronchial epithelial cells were purchased from ATCC and cultured with SAGM BulletKit (Lonza, CC-3118). Primary normal human bronchial epithelial (NHBE) cells were purchased from ATCC (PCS-300-010) and Lonza (CC-2540) and cultured with the BEGM BulletKit (Lonza, CC-3170). The air-liquid interface (ALI) differentiation was performed as we described previously.23
9
Culturing Lung Cancer and Control Cell Lines
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Lung cancer cell lines used in these experiments were purchased from the American Type Culture Collection (ATCC) and kind gifts from Jun Yokota and Takashi Kohno. Adeno 14-3 cells were established from adenocarcinoma tissues of a lung cancer patient. Further, they were cultured in RPMI-1640 (Nacalai Tesque) with 10% (vol/vol) fetal bovine serum (FBS) (ThermoFisher Scientific), 100 units/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque). SAECs were purchased from Lonza and cultured in SAGM BulletKit (Lonza). HEK293T cells (Lenti-X 293T Cell Line) were purchased from Clontech and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) supplemented with 10% (vol/vol) FBS (ThermoFisher Scientific) and 100 units/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque). All cells were cultured at 37 °C in 5% CO2.
10
Cell Culture of Lung Epithelial Lines
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BEAS-2B cells (ATCC®, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM)/Nutrient F12 Ham medium (Sigma-Aldrich, Dublin, Ireland), with 10% fetal bovine serum, penicillin G (100 U/mL), and streptomycin (100 μg/mL). The A549/NF-κB-luc cell line (Thermo Fisher, Waltham, MA) is the alveolar epithelial A549 incorporating a chromosomally integrated luciferase reporter of NF-κB transcription factor activity. These were passaged in RPMI-1640 medium (Sigma-Aldrich), with 10% fetal bovine serum, penicillin G (100 U/mL), and streptomycin (100 μg/mL). Human primary Small Airway Epithelial Cells (SAEC) (Lonza, Basel, Switzerland) were cultured in small airway cell basal medium with SAGM BulletKit (Lonza) during the expansion phase. All cell lines were cultured at 37°C in a humidified incubator with 5% CO2 in air.
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