The ER + MCF7 and T47D human breast cells, which were all obtained from ATCC, authenticated using STR profiling with CellCheck 9 Plus by IDEXX Bioresearch, and tested for mycoplasma using Mycoplasma Detection Kit, InvivoGen, Inc in April 2016, were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS; Sigma) and Antimycotic solution (Sigma). Cells were maintained in a humidified 37 °C environment with 5% CO2. pMEFs were isolated from E14.5 isogenic SIRT3+/+ mice (through a protocol that was approved by the Institutional Animal Care and Use Committee (IACUC) and complied with related animal research ethical regulations) and maintained in a 37 °C incubator with 5% CO2 and 6% oxygen, except when otherwise noted. MCF7 and T47D cells were grown for 3 months in 1 μM hydroxy-Tam to create MCF7-HTR and T47D-HTR permanent cell lines, and several different subclones were frozen. MCF7-HTR and T47D-HTR were not used for >5 passages, and new cell lines were used. All experiments were done using exponentially growing cell cultures at 50% confluence.
Antimycotic solution
Manufactured by Merck Group
Sourced in United States, United Kingdom
Antimycotic solution is a laboratory product designed to inhibit the growth of fungal organisms. It is a specialized liquid formulation that can be used in various microbiological and cell culture applications where control of fungal contamination is necessary.
Automatically generated - may contain errors
Lab products found in correlation
Gentamycin, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Nunclon delta surface sterile microtiter plates, by Thermo Fisher Scientific (2 mentions)
Edge 2.0 plate, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Mycoplasma detection kit, by InvivoGen (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Cellcheck 9 plus, by IDEXX (1 mentions)
Plasmocin prophylactic, by InvivoGen (1 mentions)
Bovine serum, by Merck Group (1 mentions)
Cpda 1, by Merck Group (1 mentions)
Fatty acid free bovine serum albumin, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Amicon ultra 15 10k, by Merck Group (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Zoletil 50, by Virbac (1 mentions)
15 protocols using antimycotic solution
1
Breast Cancer Cell Culture Protocols
2
Zika Virus Infection of Human Postnatal Thymic Epithelial Cells
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The human postnatal TEC line17 (link) was maintained in RPMI-1640 medium - supplemented with 10% fetal calf serum, 2 g/L sodium bicarbonate, 2 g/L HEPES, 1X antimycotic solution (Sigma-Aldrich) and 5 µg/mL of plasmocin prophylactic (InvivoGen) at 37 °C, in an atmosphere containing 5% CO2.
The ZIKV strain used in this study was RIO-U1, which was isolated in 2016 from urine of a patient from Rio de Janeiro state, Brazil (GenBank Accession number: KU926309), as described elsewhere49 (link). Viral stocks of ZIKV Rio-U1 were prepared by infecting Vero cell monolayers. The titer (PFU/ml) and the genome integrity were determined as described elsewhere49 (link). Samples of the stock were used in the TEC infection assays. Vero cell cultures were also applied to test the infectivity of virus particles derived from infected TEC.
Unless described elsewhere, growing human TEC was plated in T-25 flasks (105 cells) and infected 24 h later with 0.1 or 1.0 multiplicity of infection (MOI) or with 1 mL of infected TEC-derived supernatants for 1 h at 37° C in an atmosphere containing 5% CO2. After infection, culture medium was changed and cells were maintained in culture for 72 h. Controls (MOCK group) corresponded to cell grown under the same culture conditions but not infected by ZIKV.
The ZIKV strain used in this study was RIO-U1, which was isolated in 2016 from urine of a patient from Rio de Janeiro state, Brazil (GenBank Accession number: KU926309), as described elsewhere49 (link). Viral stocks of ZIKV Rio-U1 were prepared by infecting Vero cell monolayers. The titer (PFU/ml) and the genome integrity were determined as described elsewhere49 (link). Samples of the stock were used in the TEC infection assays. Vero cell cultures were also applied to test the infectivity of virus particles derived from infected TEC.
Unless described elsewhere, growing human TEC was plated in T-25 flasks (105 cells) and infected 24 h later with 0.1 or 1.0 multiplicity of infection (MOI) or with 1 mL of infected TEC-derived supernatants for 1 h at 37
3
In Vitro Hepatoprotective Assay
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The in vitro hepatoprotective assay was based on the protection of human liver-derived HepG2 cells against CCl4-induced damage. We procured HepG2 cell line from American Type Culture Collection no. (ATCC#HB-8065) (American Type Culture Collection (ATCC), Manassas, VA, USA). Human liver hepatocellular carcinoma cells (HepG2) were grown in DMEM media supplemented with 10% bovine serum, 0.1% antimycotic solution from (Sigma-Aldrich Co., St Louis, MO, USA) at 37°C in a humidified chamber with 5% CO2. The cells were then treated with medium containing 1% CCl4 along with or without the test compounds. Silymarin was used as the reference drug. Stocks of all compounds (1.0 mg/mL) were made in DMSO and further dilutions (100 µg/mL) were prepared in culture media. Cells were treated with test compounds, separately; with dose of 25 µg/mL of each compound. Treated cells were incubated in complete growth media for 48 hours. As a positive control, cells were also tested with the reference drug, silymarin. Cytotoxicity was assessed by calculating the viability of the HepG2 cells by MTT reduction assay.25 (link)
4
Bovine Blood Preparation for Testing
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Blood was collected and prepared as described previously (9 (link)). In brief, whole bovine blood was collected into 14% citrate phosphate dextrose adenine (CPDA-1) anticoagulant solution and antibiotics (50 mg/L gentamycin and 10 mL/L antimycotic solution [Sigma Aldrich, Poole, UK]) (15 ,16 ). Hematocrit levels were adjusted to 30 ± 2% by dilution with phosphate buffered saline (PBS) (Life Technologies Ltd., Paisley, UK) and transferred into the test circuit within 4 h after sample collection (15 ).
5
Isolation of Epididymal White Adipose Tissue
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Epididymal white adipose tissue (eWAT, 0.1 g) was collected and kept at room temperature in a 24-well plate with 1 ml/well of Dulbecco's modified Eagle's medium DMEM (Life Technologies, France). The tissue was minced into ~1 mm3 pieces and incubated for 1 h at 37°C and 5% CO2 prior to transferring it into a new plate with fresh DMEM medium containing 4.5 g glucose, 2 mM glutamine, 1% free fatty acid bovine serum albumin, 1% antibiotic and antimycotic solution (Sigma Aldrich). eWAT-conditioned medium (eCM) was collected 24 h after incubation and stored at −80°C for further analysis.
6
Isolation and Culture of L5 from Infected Rat Brain
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After anaesthetizing with 30 μl of Zoletil 50 (Virbac, France), living L5 were isolated from the brain tissue of infected rats. L5 were washed with saline, phosphate buffered saline (PBS), distilled water, and RPMI containing high concentration of antimycotic solution (Sigma-Aldrich, USA) before incubating in RPMI without foetal bovine serum (FBS) for 96 h. The culture medium was concentrated using Amicon Ultra-15 10 K centrifugal filter devices (Merck Millipore, Germany). The collected material was used as EPS preparations, and protein concentrations were determined with the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA).
7
Generation of Talin Knockout Cell Lines
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NIH3T3 fibroblasts and vinculin−/− mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 1% L -glutamine and 1% non-essential amino acids. For the generation of talin1 and talin2 knock out cells, cells were isolated from the collecting ducts of 5–6-weeks-old talin1flox/flox mice/ talin2 null mice56 (link) as described by Husted et al.57 and immortalized with pSV40 plasmid. Loci for talin1 gene in collecting duct cells were deleted with adenovirus expressing Cre recombinase. Multiple clones of the double null cells were prepared by performing serial dilution cloning and verified similar functions in different clones. Gene deletion was verified by both PCR and immunoblotting for talin 1 and talin 2 (Supplementary Fig. 1a ). Talin null cells were cultured in DMEM F-12 supplemented with 10% FCS, 1% L -glutamine, 15 μM HEPES and 1% Antimycotic solution (Sigma). All cells were cultured at 37 °C with 5% CO2. Transient transfections were performed using Lipofectamine and Plus reagents (Life Technologies) as per the manufacturer's instructions.
8
Preparation of Bovine Trabecular Bone Cores
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Cylindrical cores of trabecular bones explants (diameter:15 mm (0.59 in.); height:10 mm (0.39 in.)) were harvested from bovine metatarsals in a sterile manner. Briefly, the bovine metatarsals were retrieved from an abattoir on the same day of slaughter. Soft tissue was removed from the bone and a 15 mm hole saw was used to extract the core. A holding jig and a reciprocating saw were then used to cut the cores to length at 90 degrees. While cutting, the bone was continuously irrigated with 0.9% NaCl solution at 4 °C to counter heating and prevent bone debris from clogging the pores. After shaping, the bone cores were immersed in Phosphate Buffered Saline (PBS) (Sigma Aldrich, St Louis, MO) with 5% antimycotic solution (Sigma Aldrich). They then were washed twice with PBS at 37 °C for 10 min and a third time with PBS with 5% antibiotic solution for another 10 min. The cores were finally placed in a 6 well plate in 87% of DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) basal media containing l -glutamine, HEPES combined with 10% FBS and 3% antimycotic solution supplemented with 5 mM β-glycerophosphate, 5 mg/L ascorbic acid, and 0.12 g/L sodium hydrogen carbonate and placed in a humidified environment (37 °C and 5% CO2) for 24 h to acclimatize.
9
Zineb Cytotoxicity Evaluation Protocol
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Zineb (ethylene bisdithiocarbamate) was purchased from Sabero Organics (Mumbai, India). FBS, DMEM F12, antibiotics, trypsin, antimycotic solution, MTT, DCFH-DA, neutral red (NR) dye, acridine orange (AO), ethidium bromide (EtBr), PBS, propidium iodide, trypsin–EDTA solution, and cell-lysis solution were procured from Sigma-Aldrich (St Louis, MO, USA). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA). A cDNA reverse-transcription kit, SYBR green, protein standard and Trizol reagent were bought from Thermo Fisher Scientific (Waltham, MA, USA).
10
Prostate Cancer Cell Lines and Culture
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