As previously described 1 ,3 , peripheral blood eosinophils were cultured at 1×106/ml in medium, termed RPMI 10% FBS (consisting of RPMI 1640 with L-glutamine and 25 mM HEPES (Corning, Corning, NY), 10% FBS (Gibco, Thermo Fisher Scientific, Verona, WI, USA) with antibiotic/antimyotic (Gibco), 2mM L-glutamine (Gibco) and 100 mg/ml ciprofloxacin-HCL) with IL3 (4 ng/ml, R&D Systems Inc., Minneapolis, MN, USA) for 20 h. After 20 h stimulation with IL3, eosinophils were washed and suspended at 1×106/ml in fresh medium (no cytokine), and 1 ml was added to a 24-well plate, which had been coated overnight with heat-aggregated human serum immunoglobulin G (HAIgG, 10μg/ml; 500μl/well; Sigma, I-2511) and saturated with 0.1 % gelatin for 30 min at 37 °C. Heat aggregation of human IgG was performed in PBS for 30 min at 63 °C. IL3-preactivated eosinophils were also seeded on uncoated (no HAIgG) wells, as the negative control for eosinophil degranulation. After a 6 h incubation, the two types of supernatant fluids (conditioned media: IL3 and IL3 on HA-IgG (IL3-HAIgG)) were harvested and stored at −80 °C, before analysis by ELISA for EDN, IL8, and IL6 release, and before addition on cultured HBF.
Antibiotic antimyotic
Manufactured by Thermo Fisher Scientific
Sourced in United States
Antibiotic-antimyotic is a sterile solution that contains a combination of antibiotics and antifungal agents. It is used to prevent bacterial and fungal contamination in cell culture and other laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
L glutamine, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Neurobasal a medium, by Thermo Fisher Scientific (3 mentions)
Mycoalert plus mycoplasma detection kit, by Lonza (2 mentions)
High five cells, by Thermo Fisher Scientific (2 mentions)
Hek293e cells, by American Type Culture Collection (2 mentions)
Express five sef medium, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Penicillin, by Lonza (2 mentions)
Streptomycin, by Lonza (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Interleukin 3 (il 3), by Merck Group (1 mentions)
L glutamine, by Corning (1 mentions)
Interleukin 3 (il 3), by R&D Systems (1 mentions)
Hepes, by Corning (1 mentions)
Powerscan 4, by Sumitomo Pharma (1 mentions)
Ldh assay kit, by Merck Group (1 mentions)
15 protocols using antibiotic antimyotic
1
Eosinophil Degranulation Assay with IgG
2
Evaluating Cell Death and Uptake of CNTs
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The Met5A human mesothelial cell line, immortalized with SV40, was used. M199 containing 10% fetal bovine serum (#S1780, Biowest; Nuaillé, France) and 1% antibiotic-antimyotic (#15240-062, GIBCO; Grand Island, NY) was used in a humidified incubator with 5% CO2 at 37 °C. Cells were used within two months after thawing. For cell death assay, we plated cells at a density of 1×104 cells/cm2 48 h prior to the addition of each material in 96-well plates. The cell death detection assay (LDH assay kit, Sigma Aldrich; St. Louis, MO) was performed 48 h after addition of material and analyzed with a plate reader (Power Scan 4, DS Pharma Biomedical; Osaka, Japan). In the evaluation of cellular uptake, cells were observed 48 h after exposure of CNTs by phase contrast microscopy (BZ-9000, Keyence; Osaka, Japan).
3
Isolation of Neonatal Rat Cardiomyocytes
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Primary neonatal rat ventricular cardiomyocytes (NRCs) were isolated from the ventricles of 1–2-day old Sprague–Dawley rat pups as previously described [48 (link),49 (link)]; 1.5 × 106 cells per 10-cm plate were grown in DMEM (Gibco) containing 2% FBS (Gibco) and 1% antibiotic-antimyotic (Gibco). Cells were treated with or without tunicamycin 72 h post-infection. All cell culture treatments were repeated in three independent experiments.
4
Generating Gemcitabine-Resistant Pancreatic Cancer Cells
5
Influenza A Virus Cell Culture
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Raw 264.7 cells were maintained in RPMI 1640 supplemented with 10% FBS (Fetal Bovine Serum, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% antibiotic-anti-myotic (Gibco, Grand Island, NY, USA) at 37 °C, 5% CO2 in humidified air. Influenza A (A/PR/8/34) and GFP-tagged A/PR/8/34 (A/PR/8/34-GFP) viruses were used in the previous studies [15 (link)].
6
Quantifying TDP-43 Aggregation in HEK293T Cells
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HEK293T cells were from ATCC (Cat# CRL-3216 RRID:CVCL_0063 ) and have been authenticated by the vendor and were not contaminated by mycoplasma. HEK-293T cells were maintained in Dulbecco’s modified Eagle’s medium, high glucose (Gibco) containing 10% fetal bovine serum (Life Sciences), 1% non-essential amino acids (Gibco), and 1% antibiotic-antimyotic (Gibco). Cells were plated in gelatin-coated 6-well plates at a density of 3 × 106 cells/plate 24 hr before transfection. Cells were transfected with 2 µg total DNA and 7.35 µl polyethylenimine HCl MAX transfection reagent (Polysciences, Inc). Wells co-transfected with mClover3-TDPΔNLS and HSP104 variants received 1 µg of each plasmid. Media was changed 6 hr post-transfection to media containing 1 µg/ml of doxycycline hyclate (Sigma-Aldrich) to induce transgene expression. Transfected cells were lifted every 24 hr over 2 days, at which point cells were analyzed by FACS (FACSAria Fusion BD). Cells were gated to have a narrow range of FCS and SSC values and to be fluorescence positive. TDP-43 aggregation was quantified by comparing the height (FITC-H) to the width (FITC-W) of the fluorescence channel using 488 nm laser and FITC filters.
7
Culturing of Cell Lines for FcRn Studies
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HEK293E cells (ATCC, Manassas, VA, USA) and the J558L murine myeloma cell line stably producing NIP-specific hIgG1-IHH were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Sigma-Aldrich), 2 mM l -glutamine, 25 μg/ml streptomycin, and 25 U/ml penicillin (all from Bio Whittaker). The parental human microvascular endothelial cell line (WT HMEC1) and HMEC1 stably expressing HA-hFcRn-EGFP (HMEC1-hFcRn)38 (link) were grown in MCDB 131 medium (Gibco) supplemented with 10% heat-inactivated FCS, 2 mM l -glutamine and 25 µg/ml streptomycin, and 25 U/ml penicillin, 10 ng/ml mouse epidermal growth factor (PeproTech) and 1 µg/ml hydrocortisone (Sigma-Aldrich). Medium for HMEC1-hFcRn was also supplemented with 5 µg/ml blasticidin (InvivoGen) and 100 µg/ml G418 (Sigma-Aldrich) to maintain stable expression of hFcRn. High five cells (Invitrogen) were grown in Express FIVE SEF medium (Invitrogen) supplemented with 18 mM l -glutamine and 1% antibiotic–antimyotic (Invitrogen). All cell lines were negative for mycoplasma contamination (MycoAlertTM PLUS Mycoplasma detection kit, Lonza).
8
Cell Line Cultivation and Mycoplasma Testing
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High Five cells (Invitrogen) were grown in Express FIVE SEF medium (Invitrogen) supplemented with 18 mM L-glutamine and 1% antibiotic-antimyotic (Invitrogen). HEK293E cells (ATCC) were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich), 25 μg/ml streptomycin and 25 U/ml penicillin (Bio Whittaker). Both cell lines tested negative for mycoplasma contamination (MycoAlertTM PLUS Mycoplasma detection kit, Lonza).
9
Synthetic Peptide Treatment of Human Cells
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Human osteosarcoma cells (U2OS) and fibroblast (BJ-hTERT) cells were cultured in Dulbecco’s modified Eagle medium (DMEM) high glucose with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin-streptomycin at 37°C in 5% CO2. The cells were used for experiments after a one-hour treatment at 37°C with a synthetic peptide consisting of PR20 (final concentration 10 µM). Rat vascular smooth muscle cells (SMCs; Lonza, R-ASM-580) were grown in DMEM with 20% FBS and 1x Antibiotic-Antimyotic (Thermo Fisher Scientific).
10
Isolation and Culture of Human Intestinal Organoids
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Fresh tissue from human jejunum
was stripped of muscle and connective tissue using scissors to isolate
the mucosa. Small biopsies were then taken and treated with PBS +
antibiotic-antimyotic (1:100) (Thermo Scientific) for 4 × 2 min
and then PBS + DTT (10 mM) for 3 × 2 min. The tissue was then
incubated in PBS + EDTA (2 mM), 4 °C, on rotation for 1 h and
then violently shaken to isolate the crypts. The crypts were then
seeded into matrigel (hESC-Qualified Matrix, Corning) and cultured
in Intesticult (Stem Cell) to keep the cells in a stem cell state.
If growing well and forming spheres after three passages, the cultures
were considered to be stable enteroid lines and were used for further
expansion and experiments.
was stripped of muscle and connective tissue using scissors to isolate
the mucosa. Small biopsies were then taken and treated with PBS +
antibiotic-antimyotic (1:100) (Thermo Scientific) for 4 × 2 min
and then PBS + DTT (10 mM) for 3 × 2 min. The tissue was then
incubated in PBS + EDTA (2 mM), 4 °C, on rotation for 1 h and
then violently shaken to isolate the crypts. The crypts were then
seeded into matrigel (hESC-Qualified Matrix, Corning) and cultured
in Intesticult (Stem Cell) to keep the cells in a stem cell state.
If growing well and forming spheres after three passages, the cultures
were considered to be stable enteroid lines and were used for further
expansion and experiments.
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