The iNK cells were cultured in GMP Serum-free Stem Cell Growth Medium (20802, CellGenix) supplemented with 10% fetal bovine serum (FBS, 10091, Gibco). The cells were cultured at an initial density of 1×106 cells/mL. Cord blood CD34+ HSCs were usually cultured in 24-well cell culture plates at the beginning, while bone marrow CD34+ HSCs were cultured in 48- or 96-well cell culture plates and pNK cells were in 25T flasks. For CB-iNK cells and BM-iNK cells, SCF (20 ng/mL, AF−300−07, PeproTech), Flt3L (30 ng/mL, AF−300−19, PeproTech) were added to the medium during the first two weeks, and IL-15 (20 ng/mL, AF−200−15, PeproTech) was added on day 4 of culturing. At the end of day 14, the dosage of Flt3L and SCF gradually decreased until it was no longer added, while afterward only IL-15(30 ng/mL) was added from day 21. Cells were induced and cultured for approximately five weeks at 37°C in a humid atmosphere with 5% CO2. Two times a week, half of the medium was removed and replaced with fresh medium and cytokines. For the pNK-iNK group, TGF-β1 (5 ng/mL, AF−100−21C, PeproTech), hCG (10 IU/mL, LIVZON Group, China), and IL-15 (10 ng/mL) were added to the medium. Equivalent fresh medium was added after three or four days of culture, after which the plates were incubated under 1-2% O2 for the next 4~6 days.
Af 100 21c
Manufactured by Thermo Fisher Scientific
Sourced in United States
The AF-100-21C is a laboratory instrument designed for the measurement and analysis of samples. It features a compact and durable construction to withstand regular use in a laboratory environment. The core function of the AF-100-21C is to provide accurate and reliable data for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Fs22024, by Thermo Fisher Scientific (2 mentions)
Af 300 07, by Thermo Fisher Scientific (1 mentions)
Af 300 19, by Thermo Fisher Scientific (1 mentions)
Af 200 15, by Thermo Fisher Scientific (1 mentions)
Immunomagnetic separation beads, by Miltenyi Biotec (1 mentions)
Cell stimulation cocktail, by Cytek Biosciences (1 mentions)
Soluble anti cd28 antibody, by BioLegend (1 mentions)
Anti cd3 antibody, by BioLegend (1 mentions)
Af 100 11, by Thermo Fisher Scientific (1 mentions)
Af 100 20, by Thermo Fisher Scientific (1 mentions)
Af 100 15, by Thermo Fisher Scientific (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Solarbio (1 mentions)
Aml12, by American Type Culture Collection (1 mentions)
Hy 15484, by MedChemExpress (1 mentions)
Hy 12320, by MedChemExpress (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Fc 0031, by American Type Culture Collection (1 mentions)
15 protocols using af 100 21c
1
Expansion of Induced Natural Killer Cells
2
Th17 Cell Differentiation from Naive CD4+ T Cells
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Naïve CD4 cells were isolated from C57BL/6J mouse spleens using immunomagnetic separation beads (Miltenyi Biotec, 130-106-643) according to the manufacturer's instructions and seeded into 96-well plates (5 × 105/well) precoated with anti-CD3 antibody (5 μg/ml) (BioLegend, 100309) with the addition of soluble anti-CD28 antibody (2 μg/ml) (BioLegend, 102102). MSCs, sh-NC-MSCs, sh-Chi3l1-MSCs, or sh-Chi3l1-MSCs plus Stattic (20 μM, Selleck, S7024) were seeded into 96-well plates (5 × 103/well) 6 h before CD4 cell seeding. Th17 differentiation medium contained TGF-β (1.0 ng/ml) (PeproTech, AF-100-21C), IL-6 (30 ng/ml) (PeproTech, 216-16), IL-1β (20 ng/ml) (PeproTech, 211-11B), IL-23 (20 ng/ml) (BioLegend, 589002), anti-IL-4 (10 μg/ml) (BioLegend, 504102), and anti-IFN-γ (10 μg/ml) (BioLegend, 505833).
Th17 cells were differentiated for 72 h and restimulated with Cell Stimulation Cocktail (Tonbo Biosciences, TNB-4975) for 6 h before further analysis for intracellular cytokines by flow cytometry.
Th17 cells were differentiated for 72 h and restimulated with Cell Stimulation Cocktail (Tonbo Biosciences, TNB-4975) for 6 h before further analysis for intracellular cytokines by flow cytometry.
3
Defined, Serum-Free Engineered Heart Muscle
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To generate defined, serum-free EHM, purified cardiomyocytes were mixed with HFFs (American Type Culture Collection) at a 70%:30% ratio. The cell mixture was reconstituted in a mixture of pH-neutralized medical-grade bovine collagen (0.4 mg per EHM; LLC Collagen Solutions) and concentrated serum-free medium [2× RPMI, 8% B27 without insulin, penicillin (200 U/ml), and streptomycin (200 μg/ml)] and cultured for 3 days in Iscove medium with 4% B27 without insulin, 1% nonessential amino acids, 2 mM glutamine, 300 μM ascorbic acid, IGF1 (100 ng/ml; AF-100-11), FGF-2 (10 ng/ml; AF-100-18B), VEGF165 (5 ng/ml; AF-100-20), TGF-b1 (5 ng/ml; AF-100-21C; all growth factors are from PeproTech), penicillin (100 U/ml), and streptomycin (100 μg/ml) (32 (link)). After a 3-day condensation period, EHM were transferred to flexible holders to support auxotonic contractions. Analysis was carried out after a total EHM culture period of 4 weeks.
4
VEGF and TGF-β Stimulation of Cell Growth
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The cells were reseeded into 6-well plates and synchronized for 12 hours at 60% confluence. Then, the medium was replaced with H-DMEM with 2% FBS and VEGFA (5 ng/ml) or TGF-β (5 ng/ml), and cells were harvested with TRIzol after 48 hours.The cytokines used in the study were animal-free recombinant human VEGF165 (Peprotech, AF-100-20) and animal-free recombinant human TGF-β (Peprotech, AF-100-21C).
5
Optimizing Stem Cell Culture Conditions
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HSFs and OSFs were seeded at 1 × 104 cells·cm−2 in 6-well plates and cultured in a DMEM/F-12 medium supplemented with different concentrations of sodium L-ascorbate (0.25, 0.50, 0.75, and 1.0 mM), 1X Pen/Strep, and FBS, either 20% S1 1:1 S2 or 10% S2 pure, respectively. Media were changed three times a week and perforation tests were performed on 8-week-old sheets. Insulin, from an Insulin (1 g·L−1)—Transferrin (550 mg·L−1)—Selenium (670 µg·L−1) solution (Gibco #41400045), was freshly added at different concentrations (1, 2, and 4 µg·mL−1) prior to medium changes. Also growth factors, such as Epidermal Growth Factor (EGF; Peprotech #AF-100-15), Transforming Growth Factor-β1 (TGF-β1; Peprotech #AF-100-21C), basic-Fibroblast Growth Factor (bFGF; Peprotech #100-18C), and Platelet-Derived Growth Factor-BB (PDGF-BB; Peprotech #100-14B) were freshly added to the culture medium at three different concentrations (1, 3, and 9 ng·mL−1). HSFs and OSFs were seeded at the same density in 6-well plates and cultured in their respective media supplemented with 0.50 mM of sodium L-ascorbate. S2 serum contained approximately 136 ng·mL−1 of Insulin Growth Factor-1 (IGF-1) and 2.7 ng·mL−1 of Transforming Growth Factor-Beta1 (TGF-β1).22 Media were changed three times a week and perforation tests were performed on 8-week-old sheets.
6
Cultivation and Activation of Murine Hepatic Stellate Cells
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The mouse macrophage cell line RAW264.7 and mouse hepatocyte cell line AML12 were purchased from American type culture collection (ATCC; Manassas, VA, USA). The mouse HSC cell line JS-1 was purchased from Fenghbio Co., Ltd (Changsha, China). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS) (#A3161002C, Gibco) and 1% penicillin–streptomycin (#P1400, Solarbio). Primary HSCs were isolated from C57BL/6 mice according to a previously described protocol (Castello-Cros and Cukierman 2009 (link); Mederacke et al. 2015 (link)). Primary HSCs were maintained in fibroblast-specific medium (#2301, Sciencell, Carlsbad, CA, USA), and were activated by TGF-β1 (#AF-100-21C, Peprotech, Rocky Hill, NJ, USA) for 48 h before further analysis.
7
Neonatal Mouse Cardiac Fibroblast Isolation and Treatment
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Neonatal mouse cardiac fibroblasts were isolated from mice (1-2 days old). The myocardial tissue was digested in 0.25% trypsin for 2 h at 37°C. Cardiomyocytes and cardiac fibroblasts are separated based on their different adhesion times. P0 cardiac fibroblasts were cultured in DMEM medium with 10% FBS (Biological Industries, Haemek, Israel). When growing up to 70%-80% confluency, NMCFs were treated with TGF-β1 (5 ng/ml) for 6 h (PeproTech, USA, #AF-100-21C). NMCFs were also treated with 10 μM pifithrin-α, a p53 inhibitor (MedChem Express, USA, #HY-15484), or ginkgolic acid (10 μM) (MedChem Express, USA, #HY-N0077) at 1 h before TGF-β1 treatment. For the protein stability assay, 10 μg/ml cycloheximide (MedChem Express, USA, #HY-12320) was added at the indicated times.
8
Engineered Human Myocardium Protocol
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EHM was generated as described previously18 (link). In brief, iPSC-derived CMs and human foreskin fibroblasts (HFF) (HFF-1, ATCC, SCRC-1041) were mixed at a ratio 70:30, resuspended in Collagen type I (Collagen Solutions, FS22024) diluted into RPMI 2x (Thermo Fisher Scientific, 51800-035) and then cast into wells of an EHM multi-well plate (Myriamed GmbH, myrPlate-TM5). After 45 min, EHM medium freshly supplemented with TGFβ1 (Peprotech, AF-100-21C) was added, and refreshed daily for the first 3 days. Subsequently, the tissue medium was replaced daily with EHM medium for the entirety of the experimental duration.
9
Epigenetic Regulation of MRC5 Cells
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MRC5 cells, a cell line of human lung fibroblasts (BS CL 68, IZSLER-BVR, Brescia, Italy), were grown in Eagle’s minimal essential medium (MEM, Gibco, USA) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal bovine serum.
MRC5 cells were incubated in a humidified incubator at 37 °C and 5% CO to 60–80% confluence, and growth arrested for 24 h in DMEM with 0.01% FBS before adding 10 ng/ml rhTGF-β1 (AF-100-21C, Peprotech, USA) or 5 µM SAHA (CAY-10009929-100, Cayman Chemical, USA) for 24 h.
SAHA (suberoylanilide hydroxamic acid, Vorinostat, Zolinza) is a small-moleculeinhibitor of class I and class II HDACwith wide spectrum of epigenetic activities64 (link),65 (link). It has been demonstrated that SAHA inhibition of HDAC promotes cell cyclearrest, differentiation, and apoptosis of cancer cells64 (link),66 (link),67 (link). It was the first HDAC inhibitor approved by the FDA for cancer therapy.
MRC5 cells were incubated in a humidified incubator at 37 °C and 5% CO to 60–80% confluence, and growth arrested for 24 h in DMEM with 0.01% FBS before adding 10 ng/ml rhTGF-β1 (AF-100-21C, Peprotech, USA) or 5 µM SAHA (CAY-10009929-100, Cayman Chemical, USA) for 24 h.
SAHA (suberoylanilide hydroxamic acid, Vorinostat, Zolinza) is a small-moleculeinhibitor of class I and class II HDACwith wide spectrum of epigenetic activities64 (link),65 (link). It has been demonstrated that SAHA inhibition of HDAC promotes cell cyclearrest, differentiation, and apoptosis of cancer cells64 (link),66 (link),67 (link). It was the first HDAC inhibitor approved by the FDA for cancer therapy.
10
Generating Drug-Resistant and Mesenchymal Cell Lines
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Drug-resistant populations (A549R and H358R) were generated by chronical treatment (6 months) of A549 and H358 cells with stepwise incremental concentrations of MTA (0.1–1.0 µM for A549; 0.5–10 µM for H358) as previously reported31 ,39 (link). Mesenchymal populations (A549_EMT, H358_EMT), the cellular products emanating from mesenchymal reprogramming of A549 and H358 cells, were obtained after 3-month treatment (5 ng/ml for A549; 2.5 ng/ml for H358) with TGF-β1 (AF-100-21C; Peprotech, London, UK).
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