Two prostate cancer-derived cell lines (LNCaP and DU145) and one hepatocellular carcinoma-derived cell line (SNU-387), as well as a non-tumor prostate (PNT2) cell line and hepatoblastoma cell line (Hep-G2) were used in this study. All cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), except the PNT2 cells, which were generously supplied by Dr. J. De Bono (London, UK). Cells were cultured according to manufacturer instructions as previously described [82 (link),103 (link)]. Briefly, LNCaP, DU-145, PNT2, and SNU-387 cells were cultured on RPMI-1640 (Lonza, Madrid, Spain) supplemented with 10% fetal bovine serum (FBS; Merck KGaA, Damstadt, Germany), 1% L-glutamine (Thermo Fisher Scientific, Madrid, Spain), and 0.2% gentamicin-amphotericin B (Thermo Fisher Scientific), whereas Hep-G2 cells were cultured on MEM (Thermo Fisher Scientific) supplemented with 10% FBS, 1% pyruvate (Thermo Fisher Scientific), and 0.2% gentamicin-amphotericin B at 37 °C in a humidified 5% CO2 atmosphere. Cell lines were validated by analysis of short tandem repeats (STRs) sequences using GenePrint 10 System (Promega, Barcelona, Spain) and checked for mycoplasma contamination by PCR as previously reported [104 (link)].
Gentamicin amphotericin b
Manufactured by Thermo Fisher Scientific
Sourced in United States, Spain, Canada, Australia, Macao
Gentamicin/amphotericin B is a combination antibiotic solution used in cell culture media to prevent bacterial and fungal contamination. It contains the antibiotics gentamicin and amphotericin B, which target a wide range of gram-positive and gram-negative bacteria, as well as fungi.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (22 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Glutamax, by Thermo Fisher Scientific (7 mentions)
Rpmi 1640, by Lonza (5 mentions)
Dispase 2, by Roche (5 mentions)
Trypsin, by Thermo Fisher Scientific (5 mentions)
Hydrocortisone, by Merck Group (5 mentions)
Cholera toxin, by Merck Group (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (4 mentions)
Matrigel, by Corning (4 mentions)
Hepes, by Thermo Fisher Scientific (4 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Mitomycin c, by Merck Group (4 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (4 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (4 mentions)
Dmem medium, by Thermo Fisher Scientific (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Dispase, by Corning (3 mentions)
Sb202190, by Merck Group (3 mentions)
35 protocols using gentamicin amphotericin b
1
Culturing Prostate and Liver Cancer Cell Lines
2
Derivation and Culture of AVM Primary Cells
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Primary cell lines were derived from two fresh AVM tissue samples from the original cohort of 12 patients. Samples were cut into small pieces and incubated between layers of Matrigel (cat#354234, Corning Life Sciences, Tewksbury, MA, USA) in a 24-well-plate with a media containing Dulbecco's modified Eagle's medium (DMEM) with GlutaMaxTM (cat#10569010, Gibco, Rockford, IL, USA), supplemented with 2% penicillin–streptomycin (cat#15140122, Gibco), and 0.2% gentamicin/amphotericin B (cat#R01510, Gibco). Once sufficient cell growth was achieved to support transfer to a monolayer culture, cells were extracted by dissolving the Matrigel with Dispase (cat#354235, Corning Life Sciences) and transferred to an adherent culture flask with media containing DMEM with GlutaMAX™ supplemented with 10% fetal bovine serum (cat#10091148, Gibco), 5% mTeSRTM 1 Complete Medium (cat#E8580, STEMCELL Technologies, Vancouver, BC, Canada), 1% penicillin–streptomycin (cat#15140122, Gibco), and 0.2% gentamicin/amphotericin B (cat#R01510, Gibco) at 37°C and 5% CO2. Cells were expanded in culture and harvested at passages 5 and 6.
3
Optimized FACS Cell Sorting Protocol
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For fluorescence-activated cell sorting (FACS) and cytometry, cells were collected by centrifugation for 5 minutes at 500 g, re-suspended in ice-cold Sorting Medium (1% Fetal Bovine Serum in PBS, 0.25mg/mL Fungizone (Thermo Fisher Scientific), 0.25μg∕mL/10μg∕mL Amphotericin B/Gentamicin (GIBCO)) and filtered using 5mL polystyrene round-bottom tubes with cell-strainer caps (Falcon) before sorting (FACSAria) or cytometry analysis (FACSCalibur) (BD Biosciences). For sorting, cells were collected in Conditional Medium (1:1 mixture of fresh complete medium and medium collected from proliferating cell cultures that is 0.45μm filtered, supplemented with 20% Fetal Bovine Serum, 0.25mg/mL Fungizone (Thermo Fisher Scientific), 0.25μg∕mL/10μg∕mL Amphotericin B/Gentamicin (GIBCO)).
4
Airway Epithelial Cell Differentiation
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For air-liquid interface (ALI) cultures58 (link), 100,000 cultured epithelial cells per well were added to 0.4μm pore 24-well polyester membrane inserts (Corning) pre-coated with 0.03 mg/mL Type I bovine collagen solution (StemCell Technologies) with Pneumacult-Ex media (Stemcell Technologies, 05008) on both sides of the membrane. After 24 hours, apical media was changed to remove dead cells. After 72 hours, apical media was removed completely and basal media was changed to Pneumacult-ALI (Stemcell Technologies, 05001) supplemented with 5 mL 100x penicillin-streptomycin (Fisher), 1 mL 500x gentamicin/amphotericin B (ThermoFisher), 1 mL 0.2% heparin sodium salt in PBS (Stemcell Technologies) and 2.5 mL 200x hydrocortisone stock solution (Stemcell Technologies) and 0, 0.1, 1 or 10 ng/mL IL-13 (Biolegend). Basal media was changed every 2–3 days for 21 days, after which membranes were removed and cells dissociated with Stempro Accutase Cell Dissociateion Reagent (Gibco) for Seq-Well or flow cytometry. After following scRNA-seq data analysis pipelines described above, cell states recovered in ALI cultures (Fig. 5a ; Extended Data Fig. 9g ) were related to in vivo cell types59 (link).
5
Isolation and Culture of Primary cSCC Cells
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Primary cell lines were derived from 6 fresh MDHNcSCC tissue samples from the original cohort of 15 patients. The samples were cultured within a Matrigel (cat.#354234; Corning, Tewksbury, Maine) explant and extracted following their abundant growth.27 (link) The extracted cells were grown and passaged in DMEM medium (cat.#10569010; Thermo Fisher Scientific, Waltham, Mass.) supplemented with 1% fetal calf serum (cat.#10091148; Thermo Fisher Scientific), 5% mTeSR (cat.#85850; STEMCELL Technologies, Vancouver, British Columbia, Canada), 1% penicillin–streptomycin (cat.#15140122; Thermo Fisher Scientific), and 0.2% gentamicin/amphotericin B (cat.#R01510; Thermo Fisher Scientific). All cultures were maintained in a humidified 37°C incubator with 5% CO2. All primary cells used in the experiments were passages 6–8.
6
Airway Epithelial Cell Differentiation
7
Prostate Growth Medium Composition
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The human prostate growth medium included adDMEM/F12 containing 1% Penicillin/Streptomycin (v/v; Biowest; cat. no. #L0022-100), 0.2% Gentamicin/Amphotericin B (v/v; Thermo Fisher Scientific, Inc.; cat. no. #R01510), 0.2% plasmocin prophylactic (v/v; InvivoGen; cat. no. #ant-mpp), 10 mM HEPES (cat. no. #15630-056) and 2 mM GlutaMAX (cat. no. #35050-061; both Gibco; Thermo Fisher Scientific, Inc.). Components specified in Table I were added fresh on a weekly basis. For the first week after plating, ROCK inhibitor (Y-27632) was added fresh to the culture medium on the same days that medium was changed (every 2–3 days).
8
Primary Cell Lines for IHC Staining
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Primary cell lines derived from the LGCA and HGCA tissues used for IHC staining were provided by the GMRITB with approval by the Central Health and Disability Ethics Committee (Ref. 15/CEN/106). Cell lines were validated by comparison to their parent tissues via DNA sequencing [38 ]. CaCo2 (cat# HTB-37, ATCC, In Vitro Technologies, Auckland, New Zealand) cells were used as positive controls for tumorsphere formation assays. Cells were cultured in DMEM media with high glucose and containing pyruvate (cat# 10569010, ThermoFisher Scientific) and supplemented with 10% fetal calf serum (cat# 10091148, ThermoFisher Scientific), 5% mTeSR Complete (cat# 85850, STEMCELL Technologies, Tullamarine, Victoria, Australia), 1% penicillin-streptomycin (cat# 15140122, ThermoFisher Scientific) and 0.2% gentamicin/amphotericin B (cat# R01510, ThermoFisher Scientific).
9
Culturing Liver Cancer Cell Lines
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The liver cancer cell lines HepG2, Hep3B and SNU-387 (HB-8065) were used (ATCC, Manassas, USA). HepG2 and Hep3B cells were cultured in minimum essential medium (Thermo Fisher, Madrid, Spain) supplemented with 10% fetal bovine serum (FBS; Sigma‒Aldrich, Madrid, Spain), 0.2% antibiotic-antifungal (Gentamicin/amphotericin-B, Thermo Fisher) and 0.5% sodium pyruvate8 (link). SNU-387 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI-1640; Thermo Fisher) medium supplemented with 10% FBS, 0.2% antibiotic-antifungal and 0.5% glutamine (Thermo Fisher)8 (link). Cells were maintained at 37 °C and 5% CO2 under sterile conditions, periodically validated by short tandem repeat analysis (GenePrint 10 System, Promega, Barcelona, Spain) and tested for mycoplasma contamination8 (link),17 (link),18 (link). Positive and/or negative controls [IGF-1 (10-6 M) and paclitaxel (10-7 M), respectively] were used.
10
Culturing Primary Cell Lines from Colorectal Cancer
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Primary cell lines derived from 3 low-grade (LG) and 3 high-grade (HG) CA tissue samples included in our previous study [16 (link)] were provided by the Gillies McIndoe Research Institute Tissue Bank for this study with approval by the Central Health and Disability Ethics Committee (Ref. 15/CEN/106). Written consent was obtained from all participants. Commercial cell lines were used as positive controls for tumorsphere formation (CaCo2; cat# HTB-37, ATCC, In Vitro Technologies, Auckland, New Zealand), differentiation and α-SMA expression (3T3; cat# CRL-1658, ATCC), EpCAM expression (HepG2; cat# HB8025, ATCC) and iPSC marker expression (NTERA-2; cat# CRL-1973, ATCC).
Cells were cultured in Nunc™ EasYFlasks™ (Thermo Fisher Scientific, Waltham, MA, USA) using DMEM media with high glucose concentration and containing pyruvate (cat# 10569010, Thermo), and supplemented with 10% fetal calf serum (FCS; cat# 10091148, Thermo), 5% mTeSR Complete (cat# 85850, STEMCELL Technologies, Tullamarine, VIC, Australia), 1% penicillin/streptomycin (cat# 15140122, Thermo) and 0.2% gentamicin/amphotericin B (cat# R01510 Thermo).
Cells were passaged upon reaching 75–95% confluency using PBS (cat# 70013032, Thermo) to wash the cells and TrypLE Express Enzyme (cat# 12605093, Thermo) to detach them from the flask.
Cells were cultured in Nunc™ EasYFlasks™ (Thermo Fisher Scientific, Waltham, MA, USA) using DMEM media with high glucose concentration and containing pyruvate (cat# 10569010, Thermo), and supplemented with 10% fetal calf serum (FCS; cat# 10091148, Thermo), 5% mTeSR Complete (cat# 85850, STEMCELL Technologies, Tullamarine, VIC, Australia), 1% penicillin/streptomycin (cat# 15140122, Thermo) and 0.2% gentamicin/amphotericin B (cat# R01510 Thermo).
Cells were passaged upon reaching 75–95% confluency using PBS (cat# 70013032, Thermo) to wash the cells and TrypLE Express Enzyme (cat# 12605093, Thermo) to detach them from the flask.
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