hPSCs were dissociated by Accutase (Sigma) and plated on growth factor-reduced Matrigel (Corning)-coated plates with thiazovivin (0.1 μM, Selleck). hPSCs were induced for stepwise blood differentiation in basal medium supplemented with cytokines and inhibitors.23 (link) Briefly, hPSCs were treated with DMEM/F12 (Gibco) medium containing 40 ng/mL BMP4 (Peprotech), 30 ng/mL ACTIVIN A (Sino Biological), 20 ng/mL basic fibroblast growth factor (bFGF) (Sino Biological), 6 μM CHIR99021 (Selleck), and 10 μM LY294002 (Selleck) for 1–2 days. Then, we switched to medium containing 40 ng/mL vascular endothelial growth factor (Sino Biological) and 50 ng/mL bFGF for 2 or 3 days. HSPCs were induced by medium containing 10 ng/mL SCF (Peprotech), 50 ng/mL thrombopoietin (Sino Biological), 10 ng/mL IL-3 (Sino Biological), 50 ng/mL IL-6 (Sino Biological), and 50 ng/mL FMS-like tyrosine kinase 3 ligand (FLT3L) (Peprotech). Floating iHSPCs were collected and plated in myeloid differentiation medium (StemPro +50 ng/mL FLT3L + 50 ng/mL M-CSF + 25 ng/mL GM-CSF) for 12 days and switched to macrophage maturation medium (RPMI-1640 + 10% FBS + 50 ng/mL M-CSF) for 7 days. All of the recombinant human cytokines were purchased from Sino Biological.
Basic fibroblast growth factor (bfgf)
Manufactured by Sino Biological
Sourced in China, United States, Israel
BFGF is a recombinant human basic fibroblast growth factor (bFGF) protein. bFGF is a heparin-binding growth factor involved in various cellular processes, including cell growth, differentiation, and angiogenesis.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by Sino Biological (9 mentions)
B27 supplement, by Thermo Fisher Scientific (7 mentions)
Sb431542, by Selleck Chemicals (7 mentions)
Vascular endothelial growth factor (vegf), by Sino Biological (6 mentions)
Bone morphogenetic protein 4 (bmp4), by Thermo Fisher Scientific (6 mentions)
Chir99021, by Selleck Chemicals (5 mentions)
Activin a, by Sino Biological (5 mentions)
Dmem f12, by Thermo Fisher Scientific (4 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (4 mentions)
Ly294002, by Selleck Chemicals (4 mentions)
Heparin, by Merck Group (3 mentions)
Insulin, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Hydrocortisone, by Merck Group (3 mentions)
Thiazovivin, by Selleck Chemicals (3 mentions)
Thrombopoietin, by Sino Biological (3 mentions)
Flt3l, by Thermo Fisher Scientific (3 mentions)
Interleukin 3 (il 3), by Sino Biological (3 mentions)
Interleukin 6 (il 6), by Sino Biological (3 mentions)
Accutase, by Merck Group (3 mentions)
28 protocols using basic fibroblast growth factor (bfgf)
1
Stepwise Differentiation of hPSCs to Macrophages
2
Directed Hematopoietic Differentiation of hPSCs
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Before haematopoietic differentiation, hPSCs were dissociated by Accutase (Sigma) and plated on growth factor‐reduced Matrigel (Corning)‐coated plates with thiazovivin (0.1 μM, Selleck). Firstly, at day 0, 40 ng/ml of BMP4 (Peprotech), 30 ng/ml of ACTIVINA (Sino Biological Inc.), 20 ng/ml of bFGF (Sino Biological Inc.), 6 μM CHIR99021 (Selleck) and 10 μM LY294002 (Selleck) were added to the basic medium (BM, mimics of the CustommTeSR1) of Dulbecco's‐modified Eagle's medium/F‐12 (GIBCO) supplemented with 1% insulin–transferrin–selenium (GIBCO), 70 μg/ml of vitamin C (Sigma). Second, 30 ng/ml of BMP, 1 μM A8301 (Selleck) and 2 μM IWR‐1‐endo (Selleck) were added to the BM on day 1. Then, on days 2–4 of differentiation, 40 ng/ml of vascular endothelial growth factor (Sino Biological Inc.) and 50 ng/ml of bFGF were added to the BM. Finally, 40 ng/ml of vascular endothelial growth factor, 50 ng/ml of bFGF, 10 μM SB431542 (Selleck), 10 ng/ml of stem cell factor (Peprotech), 50 ng/ml of thrombopoietin (Sino Biological Inc.), 10 ng/ml ofinterleukin 3 (Sino Biological Inc.) and 50 ng/ml of interleukin 6 (Sino Biological Inc.) were added in the BM at days 4–6 of differentiation and further haematopoietic commitment and maturation.
3
Hepatic Differentiation of Cord-Derived MSCs
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The third passage cultured cells were harvested at a density of 1 × 105 cells/well until 70%-80% confluence was reached and then seeded in DMEM/F12 medium supplemented with 20 ng/mL epidermal growth factor (EGF, Sino Biological, China) and 10 ng/mL basic fibroblast growth factor (bFGF, Sino Biological, China) for two days. To induce hepatic differentiation, this study drew upon the previously reported three-step protocol designed for human umbilical cord-derived MSCs [18 (link)]. The step-1 induction medium consists of DMEM/F12 supplemented with 20 ng/mL hepatocyte growth factor (HGF, Sino Biological), 10 ng/mL bFGF (Sino Biological, China), and 0.61 g/L nicotinamide (NTA, Sigma-Aldrich, USA) for 7 days. Thereafter, the cells were treated by a maturation medium including DMEM/F12 supplemented with 20 ng/mL oncostatin M (OSM, Sino Biological, China), 1 μmol/L dexamethasone (Dexa, Solarbio, China), and insulin-transferrin-selenium premix (ITS, Sigma-Aldrich, USA) for an additional week. The culture medium was replaced with fresh medium twice a week.
4
Sphere Formation Assay in Serum-Free
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Nonadherent dishes (Costar, Corning, NY, USA) were used for this assay. Cells were plated and maintained in serum-free medium (SFM) consisting of DMEM-F12 (Gibco, Carlsbad, CA, USA), 30 ng/mL EGF (Sino Biological; #10605), 15 ng/mL basic fibroblast growth factor (Sino Biological; #10014), 10 μg/mL insulin (Sino Biological; #11038), 0.4% BSA (Sigma-Aldrich, St. Louis, MO, USA), and 2% B27 (Gibco, Carlsbad, CA, USA). Photographs were obtained and the number of spheres assessed by microscopy (Nikon, Tokyo, Japan).
5
Isolation of Neural Crest Stem Cells
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Fresh human skin tissue was harvested and then stored in ice-cold PBS. Neural crest stem cells were isolated using a previously published method (Krejci and Grim, 2010; Yu et al., 2010). Briefly, after several PBS washes and careful scraping of attached adipose tissue, skin tissue was cut into small blocks and then treated with Dulbecco's Modified Eagle's Medium (DMEM) containing 4.8 mg/mL dispase (Invitrogen, Grand Island, NY, USA) at 4°C overnight. Under a light microscope (Olympus, Shanghai, China), hair follicles were isolated from deciduous epidermis, collected and then washed with PBS. The hair follicles were treated twice with 0.25% trypsin/ethylenediamine tetraacetic acid (Invitrogen) for 30 minutes each. The resultant cell suspension was filtered with a 40-μm cell strainer (BD Falcon, Bedford, MA, USA). After counting, cells were seeded in a Petri dish, in which human embryonic stem cell medium (Gibco, Shanghai, China) containing 4 ng/mL basic fibroblast growth factor (Sino Biological, Beijing, China) was added. Cells were then incubated in a 5% CO2 incubator at 37°C. Culture medium was renewed once every 3 days.
6
Mammosphere Cultivation and Passage
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Mammopshere cultivation was performed as previously described [41 , 42 (link)]. Briefly, Cells were suspended into DMEM/F12 medium supplemented with 0.4% bovine serum albumin (Sigma-Aldrich), 10 ng/ml epidermal growth factor (PeproTech Asia, Rehovot, Israel), 10 ng/ml basic fibroblast growth factor (Sino Biological Inc., Beijing, China), 0.5X B27 supplement (Thermo Fisher Scientific, Waltham, MA, USA), 5 μg/ml insulin (Sigma-Aldrich), 1 μg/ml hydrocortisone (Sigma-Aldrich) and 4 μg/ml heparin (Sigma-Aldrich) and seeded into ultralow attachment 6-well-plate (Greiner Bio-One GmbH, Kremsmünster, Austria) at a density of 1×104/ml. The number of formed primary mammospheres with a diameter larger than 50 μm was counted under an inverted microscopy. Primary mammospheres were harvested by filtering with 70 μm cell strainer (BD Biosciences), dissociated into single cell suspension by HyQTase and performed secondary mammosphere formation as the protocol of primary mammosphere cultivation.
7
Mammosphere Formation Assay for Breast Cancer
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For primary mammosphere formation, AS-B145 or BT-474 cells were suspended as 1 × 104/well/2 mL and cultured in ultralow attachment 6-well-plates (Corning, Lowell, MA, USA) in DMEM/F12 medium supplemented with 0.4% bovine serum albumin (Sigma), 20 ng/mL EGF (PeproTech, Rocky Hill, NJ, USA) , 20 ng/mL basic fibroblast growth factor (Sino Biological Inc., Beijing, China), 4 μg/mL heparin (Sigma-Aldrich), 5 μg/mL insulin, 1 μM hydrocortisone (Sigma-Aldrich), and 1X B27 supplement (Gibco). After 7 days, primary mammospheres with diameter larger than 50 μm were collected with 70 μm cell strainer (BD Biosciences, San Jose, CA, USA) and dissociated into single cell suspension with HyQTase (GE Healthcare Life Sciences HyClone Laboratories, Logan, UT, USA) for secondary mammosphere formation at a cell density as 2500 cells/well/2mL for further seven days. The form secondary mammospheres were pictured and counted with an inverted microscopy (AE30, Motic Electric Group Co., Ltd., Xiamen, China).
8
Isolation and Cultivation of HF-MSCs
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The isolation and identification of HF-MSCs were carried out as in our previous study [15 (link), 20 (link)]. Isolated HF-MSCs were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 2 ng/mL of basic fibroblast growth factor (Sino Biological Inc., China) and 100 U/mL penicillin–streptomycin (Hyclone). HEK 293 T cells were cultured in DMEM containing 10% FBS and 100 U/mL penicillin–streptomycin. Cells were cultured at 37 °C and 5% CO2.When the cells proliferated to an 80%–90% confluence, they were digested and subcultured under similar individual conditions.
9
Isolation and Culture of HF-MSCs
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This study was approved by Ethnic Committee of School of Public Health, Jilin University (2021-06-06). The isolation and identification of HF-MSCs were performed in our previous study (Jiang et al., 2019 (link); Liu et al., 2019 (link); Shi et al., 2019 (link)). HF-MSCs were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, United States ) containing 10% fetal bovine serum (FBS, Hyclone, United States ), 2 ng/ml basic fibroblast growth factor (Sino Biological Inc., China) and 100 U/ml penicillin-streptomycin (Hyclone, Logan, UT, United States ). HEK293T cells were cultured in DMEM containing 10% FBS and 100 U/ml penicillin-streptomycin. The cells were cultured at 37°C and 5% CO2. When HF-MSCs or HEK293T proliferated to 80% confluence, they were digested with 0.25% trypsin and subcultured under the same individual conditions.
10
Spheroid Formation from Hepatocellular Carcinoma
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Hepatocellular carcinoma cells were plated in 96‐well ultra‐low attachment culture dishes (Cat# 3474; Corning) at 200 cells per well and incubated in DMEM/F12 medium (Cat# C11330500BT; Gibco) supplemented with 20 ng/ml basic fibroblast growth factor (Cat# HG10014‐NH; Sino Biological) and 20 ng/ml epidermal growth factor (Cat# HG10325‐M; Sino Biological), 2% B27 supplement (Cat# 17504–044; Thermo Fisher Scientific), for 7 days. The number of spheroids was counted, and representative views were imaged using a microscope (Cat# 135706; Nikon Corporation).
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