The H492-dys Ex31m, H492-dys Ex31w, and H492-dys Ex27m plasmids were prepared as described previously8 (link). HeLa cells were maintained in low-glucose Dulbecco’s modified Eagle’s medium (Nacalai Tesque) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Immortalized muscle cells (D4P4) from a DMD patient with the c.4303G > T mutation were established as described previously28 (link). Cells were maintained in Dulbecco’s modified Eagle’s medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 20% fetal bovine serum (Gibco, Grand Island, NY, USA), 2% Ultroser™ G serum substitute (Pall Corp., NY, USA), and 1% antibiotic-antimycotic solution (Gibco). Transfections were carried out using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s instructions. For reporter assays, TG693 and TG003 were added to the culture medium for 24 h before analysis.
Ultroser g serum substitute
Manufactured by Pall Corporation
Sourced in United States, Canada
Ultroser G is a serum substitute for cell culture applications. It is a protein-rich, chemically defined, and low-endotoxin formulation derived from bovine serum. Ultroser G is designed to support cell growth and proliferation in a variety of cell culture systems.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Catalog 7060, by ScienCell (2 mentions)
Ultroser g serum substitute, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Prigrow 4 medium, by Applied Biological Materials (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Nacalai Tesque (1 mentions)
Dulbecco s modified eagle s medium, by Nacalai Tesque (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Fujifilm (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Mcdb105 medium, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by System Biosciences (1 mentions)
Angiotensin 2, by Merck Group (1 mentions)
Six well collagen coated plates, by Corning (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Corning (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
Mlpenicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
15 protocols using ultroser g serum substitute
1
Plasmid Preparation and Cell Culture Protocol
2
Immortalized Fallopian Tube Secretory Epithelial Cell Culture
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Immortalized human fallopian tube secretory epithelial cell line (FTSEC) was kindly provided by Dr. Ronny Drapkin [12 (link)]. Ovarian surface epithelial (OSE7, HOSE1–15) and high-grade serous ovarian cancer (SKOV3, OVCA3) cell lines have been previously described [13 ]. FTSEC cells were cultured in DMEM/Ham’s F-12 1:1 (Cellgro, Mediatech, Inc. Manassas, VA) supplemented with 2% Ultroser G serum substitute (Pall Corp., Port Washington, NY). Ovarian epithelial cell lines were cultured in a mixture of medium 199 and MCDB105 medium (1:1) (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA).
Standard media was replaced with exosome-free fetal bovine serum (System Biosciences, SBI, Palo Alto, CA) containing media 24 h prior to conditioned media collection. Cell counts were determined at the time of conditioned media collection and ranged from 2 to 9 × 105 cells/mL. Conditioned media were transferred to 50 cm3 tubes and centrifuged at 2000×g for 15 min at 4 °C to remove cells and cell debris. The supernatant was then stored at − 80 °C until further use.
Standard media was replaced with exosome-free fetal bovine serum (System Biosciences, SBI, Palo Alto, CA) containing media 24 h prior to conditioned media collection. Cell counts were determined at the time of conditioned media collection and ranged from 2 to 9 × 105 cells/mL. Conditioned media were transferred to 50 cm3 tubes and centrifuged at 2000×g for 15 min at 4 °C to remove cells and cell debris. The supernatant was then stored at − 80 °C until further use.
3
Cervical Epithelial Cell Culture and Genetic Manipulation
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Primary hCerECs were cultured following a protocol provided by the vendor (Catalog #7060, ScienCell Research Laboratories, Inc.). SiHa (a human cervical cancer cell line contained HPV16) and ECT1 (a human endocervical cell line immortalized by HPV16 E6/E7) cells were cultured with 2% Ultroser G serum substitute (Pall Corporation) in DMEM/F12 medium. hCerEC-MX and ECT1-MX cells was generated by transfecting hCerEC and ECT1 cells with retrovirus-based empty MXIV vector (MX) as control; hCerEC-YAP and ECT1-YAP cells were generated by transfecting hCerEC and ECT1 cells with vectors expressing wild-type of YAP1 protein; hCerEC-YAPS127A and ECT1-YAPS127A cells were generated by transfecting hCerEC and ECT1 cells with vectors expressing constitutively active YAP1 (YAPS127A, Serine at residue 127 is replaced by an Alanine resulting in the constitutive activation of YAP1). Since hCerEC are primary cells, all experiments using hCerEC-MX, hCerEC-YAP and hCerEC-YAPS127A cells were performed within 4 passages after transfection. Expression of YAP1 gene was confirmed by both RT-PCR and western blot assay.
4
Angiotensin II Treatment of Human Granulosa Cells
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The human granulosa cell line (hGL5) was purchased from Applied Biological Materials (Richmond, BC, Canada) and cultured in Prigrow IV medium (Applied Biological Materials), supplemented with 1× Insulin-Transferrin-Selenium (ITS; Zen-Bio, Durham, NC, USA), 4% Ultroser G serum substitute (Pall Corporation, Port Washington, NY, USA), 2% calf serum (Hyclone, Logan, UT, USA), and 1% penicillin/streptomycin (P/S; Gibco), according to the manufacturers’ instructions. The hGL5 cells (2 × 104/well) were seeded in six-well collagen-coated plates (Corning, NY, USA) for 24 h and treated with 0–100 µM angiotensin II (Sigma-Aldrich) for 24 h in a low-serum-culture medium containing 1× ITS, 0.4% Ultroser G serum substitute, and 1% P/S. The cells were then collected from the culture medium for flow cytometry analysis.
5
Immortalized Fallopian Tube Cell Lines
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FT194 (human TERT, SV40 large T antigen) and FT246 (human TERT, TP53-shRNA, human CDK4.R24C overexpression) fallopian tube secretory epithelial cell lines were generously provided by Dr. R. Drapkin (University of Pennsylvania, USA). Cells were cultured in DMEM/F12 medium (Gibco) supplemented with 2% Ultroser G serum substitute (Pall Corporation, Pall France) and maintained at 37 °C in a humidified incubator (5% CO2).
6
4HC Treatment of hGL5 Cells
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The hGL5 cell line was purchased from Applied Biological Materials (Richmond, BC, Canada) and cultured in Prigrow IV Medium (Applied Biological Materials) supplemented with 1× ITS, 4% Ultroser G Serum Substitute (Pall Corporation, Port Washington, NY), 5% calf serum (Hyclone, UT) and 1% penicillin/streptomycin (P/S, Gibco) according to the manufacturers' instructions. Cells (2 × 104/well) were seeded in 6‐well collagen‐coated plates (Corning, Corning, NY) for 24 h. The cells were treated with 0 or 0.5 μg/ml 4HC (Sigma‐Aldrich) in culture media for 4 h, then washed and cultured with low serum medium containing 1× ITS, 0.4% Ultroser G Serum Substitute, and 1% P/S. Cells were collected in Cell Staining Buffer for flow cytometry immediately after 4HC treatment and 24 h later.
7
Cell Culture Protocols for Various Cell Lines
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MOE cells were maintained in αMEM
(10-022-CV, Cellgro, Manassas, VA) supplemented with 10% FBS (FB5001,
Denville Scientific, Holliston, MA), 2 mMl -glutamine (30005068,
CellGro, Manassas, VA), 10 mg/mL ITS (11074547001, Roche, Indianapolis,
IN), 1.8 ng/mL EGF (100-15, Peprotech Inc., Rocky Hill, NJ), 100 U/mL
penicillin–streptomycin (15140-122, Gibco, Grand Island, NY),
1 mg/mL gentamycin (30-005-CR, CellGro, Manassas, VA), and 18.2 ng/mL
estradiol-17β (E1024-1G, Sigma-Aldrich, St. Louis, MO). MOE
SCRshRNA, PTENshRNA, and p53R273H were maintained in similar media but with selection antibiotic.
MOSE cells were maintained in media similar to MOE cells except estradiol
was not added. FT33 Myc cells were maintained in DMEM/F-12 media (11330-032,
Thermo Scientific, Waltham, MA) supplemented with 2% Ultroser G serum
substitute (15950-01, Pall Corporation, Port Washington, New York).
SKOV3 cells were maintained in McCoy’s 5A (LT 16600-082, Life
Technologies) supplemented with 1.1 g of sodium bicarbonate per 500
mL, and 10% FBS. Cells were passaged every 3–4 days and incubated
at 5% CO2 and 37 °C.
(10-022-CV, Cellgro, Manassas, VA) supplemented with 10% FBS (FB5001,
Denville Scientific, Holliston, MA), 2 mM
CellGro, Manassas, VA), 10 mg/mL ITS (11074547001, Roche, Indianapolis,
IN), 1.8 ng/mL EGF (100-15, Peprotech Inc., Rocky Hill, NJ), 100 U/mL
penicillin–streptomycin (15140-122, Gibco, Grand Island, NY),
1 mg/mL gentamycin (30-005-CR, CellGro, Manassas, VA), and 18.2 ng/mL
estradiol-17β (E1024-1G, Sigma-Aldrich, St. Louis, MO). MOE
SCRshRNA, PTENshRNA, and p53R273H were maintained in similar media but with selection antibiotic.
MOSE cells were maintained in media similar to MOE cells except estradiol
was not added. FT33 Myc cells were maintained in DMEM/F-12 media (11330-032,
Thermo Scientific, Waltham, MA) supplemented with 2% Ultroser G serum
substitute (15950-01, Pall Corporation, Port Washington, New York).
SKOV3 cells were maintained in McCoy’s 5A (LT 16600-082, Life
Technologies) supplemented with 1.1 g of sodium bicarbonate per 500
mL, and 10% FBS. Cells were passaged every 3–4 days and incubated
at 5% CO2 and 37 °C.
8
Synthesis and Characterization of Iron Oxide Nanoparticles for Cell Culture
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All chemicals were of analytical reagent grade and were used without further purification. Deionized water was used to synthesize the materials. Iron(III) chloride hexahydrate (FeCl3·6H2O), hydrochloric acid, and ethanol were purchased from Sinopharm Chemical Reagent Corporation, Ltd. Bovine serum albumin (BSA) and Triton X‐100 were purchased from Solarbio (Beijing, China), and paraformaldehyde solution was purchased from SparkJade (Qingdao, China).
For cell experiments, the ultraCULTURE serum‐free culture medium was purchased from Lonza Bioscience (USA), and the Ultroser G serum substitute was purchased from Pall Corporation (USA). Fetal bovine serum (FBS) was purchased from Gibco (USA). Basic fibroblast growth factor (bFGF) was purchased from Peprotech (USA). Penicillin‐streptomycin solution was purchased from SparkJade (China). The CCK‐8 kit was purchased from MedChemExpress (Shanghai, China), and Trizol reagent was purchased from Invitrogen (USA). The primers for nestin, Tuj1, GFAP, and MAP2 for qPCR were purchased from Sangon Biotech (Shanghai, China). 4’6‐diamidino‐2‐phenylindole (DAPI), nestin, Tuj1, GFAP, and MAP2 primary and secondary antibodies were purchased from Abcam (USA).
For cell experiments, the ultraCULTURE serum‐free culture medium was purchased from Lonza Bioscience (USA), and the Ultroser G serum substitute was purchased from Pall Corporation (USA). Fetal bovine serum (FBS) was purchased from Gibco (USA). Basic fibroblast growth factor (bFGF) was purchased from Peprotech (USA). Penicillin‐streptomycin solution was purchased from SparkJade (China). The CCK‐8 kit was purchased from MedChemExpress (Shanghai, China), and Trizol reagent was purchased from Invitrogen (USA). The primers for nestin, Tuj1, GFAP, and MAP2 for qPCR were purchased from Sangon Biotech (Shanghai, China). 4’6‐diamidino‐2‐phenylindole (DAPI), nestin, Tuj1, GFAP, and MAP2 primary and secondary antibodies were purchased from Abcam (USA).
9
Cervical Epithelial Cell Culture and Genetic Manipulation
10
Melanocyte Isolation and Culture
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