HB-EGF, HGF, FGF-2, VEGF, SDF-1, FGF-2, human recombinant VEGF165, stromal cell-derived factor-1 were purchased from R&D Systems (Minneapolis, MN). PG545 was synthesized by Progen Pharmaceuticals (Brisbane, QLD, Australia). All drugs (PG545, cisplatin (Calbiochem, Millipore, Billerica, MA), carboplatin (50mg/5ml, Novaplus, Novation, Irvine, TX), paclitaxel (30mg/5ml, Hospira, Lake Forest, IL) were dissolved using cell culture medium for in vitro experiments and phosphate buffered saline (PBS) for in vivo studies.
Fibroblast growth factor 2 (fgf2)
Manufactured by R&D Systems
Sourced in United States, United Kingdom, Germany, Canada, China
FGF2 is a recombinant human protein that functions as a fibroblast growth factor. It is involved in the regulation of cell growth, differentiation, and angiogenesis.
Automatically generated - may contain errors
Lab products found in correlation
Epidermal growth factor (egf), by R&D Systems (41 mentions)
Dmem f12, by Thermo Fisher Scientific (40 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (37 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (30 mentions)
Glutamax, by Thermo Fisher Scientific (30 mentions)
L glutamine, by Thermo Fisher Scientific (29 mentions)
Activin a, by R&D Systems (24 mentions)
B27 supplement, by Thermo Fisher Scientific (23 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (23 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (20 mentions)
N2 supplement, by Thermo Fisher Scientific (18 mentions)
Neurobasal medium, by Thermo Fisher Scientific (17 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (16 mentions)
Heparin, by Merck Group (15 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (15 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (15 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (13 mentions)
Vascular endothelial growth factor (vegf), by R&D Systems (12 mentions)
β mercaptoethanol, by Merck Group (12 mentions)
Streptomycin, by Thermo Fisher Scientific (12 mentions)
345 protocols using fibroblast growth factor 2 (fgf2)
1
Cytokine and Drug Treatments Protocol
2
Directed Differentiation of hiPSCs into Endoderm and Mesoderm
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hiPSC cells grow in mTeSR were dissociated into single cells using accutase and plated at density of 25–50 k cells/cm2 on matrigel coated cell culture plates and subsequently differentiated into endoderm and mesoderm lineages (Loh et al., 2014 (link); Ang et al., 2018 (link)). For definitive endoderm induction, anterior primitive streak was first specified using 100 ng/ml Activin A (R and D systems, 338-AC-050), 3 μm CHIR (Tocris, 4423) and 20 ng/ml FGF2 (R and D Systems, 233-FB-01M) in CDM2 basal media. After 24 hr, the cells were washed with DMEM/F12 (1x, Thermo Fisher Scientific, 11320033), and definitive endoderm was induced using 100 ng/ml Activin A and 250 nM LDN (Reprocell, Yokohama, Japan; 04–0074) in CDM2 basal media for 24 hr. For lateral mesoderm induction, midprimitive streak was specified using 30 ng/ml Activin, 16 μM CHIR, 20 ng/ml FGF2 and 40 ng/ml BMP (R and D Systems, 314 BP-050) in CDM2 basal media for 24 hr. After 24 hr, cells were washed with DMEM/F12 (1x, Thermo Fisher Scientific, 11320033), and lateral mesoderm was induced using 1 μM A8301 (R and D Systems, 2939) 30 ng/ml BMP and 1 μM C59 (Tocris, Bristol, United Kingdom; 5148) for 24 hr in CDM2 basal media. On the third day, cells were lysed for RNA collection and purification.
3
C6 Glioma Cell Culture and Stimulation
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C6 glioma
cells were cultured with the same protocol as for NSPCs, on plates
precoated with poly-l -ornithine and fibronectin, and then
grown in N2 medium with supplements. The cells were plated in a 6-well
plate (40,000 cells/well) in serum-free DMEM:F12 (Invitrogen) with
supplements and 10 ng/mL FGF2 (R&D systems) for 24 h prior to
further stimulation. Cells were then stimulated with 10 ng/mL FGF2,
10 ng/mL CNTF (R&D systems), 10% FBS (Invitrogen), 1 mM VPA (Sigma),
or without added factors (N2 medium) for 3 days. Addition of soluble factors was carried out
every 24 h, and media was changed every 48 h. p-HTMI was administered,
from a stock solution of 1 mg/mL at a dilution of 1:500, directly
to each well, and the detection was done after 10 min in a fluorescent
microscope. p-HTMI generated chemoluminescence at a wavelength common
to green fluorescent proteins.
cells were cultured with the same protocol as for NSPCs, on plates
precoated with poly-
grown in N2 medium with supplements. The cells were plated in a 6-well
plate (40,000 cells/well) in serum-free DMEM:F12 (Invitrogen) with
supplements and 10 ng/mL FGF2 (R&D systems) for 24 h prior to
further stimulation. Cells were then stimulated with 10 ng/mL FGF2,
10 ng/mL CNTF (R&D systems), 10% FBS (Invitrogen), 1 mM VPA (Sigma),
or without added factors (N2 medium) for 3 days. Addition of soluble factors was carried out
every 24 h, and media was changed every 48 h. p-HTMI was administered,
from a stock solution of 1 mg/mL at a dilution of 1:500, directly
to each well, and the detection was done after 10 min in a fluorescent
microscope. p-HTMI generated chemoluminescence at a wavelength common
to green fluorescent proteins.
4
Differentiation of Neural Stem Cells
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NSPCs were plated in a 6-well
plate (40,000 cells/well) in serum-free DMEM:F12 (Invitrogen) with
supplements and 10 ng/mL FGF2 (R&D systems) for 24 h prior to
further stimulation. Cells were then stimulated with 10 ng/mL FGF2,
10 ng/mL CNTF (R&D systems), 10% FBS (Invitrogen), 1 mM VPA (Sigma),
or without added factors (N2 medium) for 3 days. Addition of soluble
factors was carried out every 24 h, and media was changed every 48
h. The LCOs were dissolved in deionized water to a final concentration
of 1 mg/mL and administered at a dilution of 1:500, directly to each
well, and the detection was done after 10 min in a fluorescent microscope.
p-HTMI generated fluorescence at a wavelength common to green fluorescent
proteins. A strong green signal was obtained in undifferentiated immature
stem cells, accumulated in the cytoplasm of the cells, whereas differentiated
and more mature cells displayed a significantly lower or no signal.
Neural stem cells were also treated with 10 ng/mL BMP4 and 10 ng/mL
Wnt3a (R&D Systems) for 14 days and then stained with p-HTMI.
plate (40,000 cells/well) in serum-free DMEM:F12 (Invitrogen) with
supplements and 10 ng/mL FGF2 (R&D systems) for 24 h prior to
further stimulation. Cells were then stimulated with 10 ng/mL FGF2,
10 ng/mL CNTF (R&D systems), 10% FBS (Invitrogen), 1 mM VPA (Sigma),
or without added factors (N2 medium) for 3 days. Addition of soluble
factors was carried out every 24 h, and media was changed every 48
h. The LCOs were dissolved in deionized water to a final concentration
of 1 mg/mL and administered at a dilution of 1:500, directly to each
well, and the detection was done after 10 min in a fluorescent microscope.
p-HTMI generated fluorescence at a wavelength common to green fluorescent
proteins. A strong green signal was obtained in undifferentiated immature
stem cells, accumulated in the cytoplasm of the cells, whereas differentiated
and more mature cells displayed a significantly lower or no signal.
Neural stem cells were also treated with 10 ng/mL BMP4 and 10 ng/mL
Wnt3a (R&D Systems) for 14 days and then stained with p-HTMI.
5
Definitive Endoderm Differentiation of hPSCs
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The procedure for definitive endoderm differentiation of hPSCs was based on a previously described protocol (Rashid et al., 2010 (link); Hannan et al., 2013 (link)). In brief, for initiation of definitive endoderm differentiation, hPSCs were detached with EDTA (15575–020; Thermo Fisher Scientific), filtered through a 70-µm filter (352350; Corning) and seeded as small clumps in E8 media with 10 µM Y27632 (Y0503; Sigma-Aldrich) on gelatin-coated plates preincubated overnight with mouse embryonic fibroblast media. Differentiation was started 48 h later by changing media to day 1 media, consisting of CDM-PVA media + 100 ng/ml Activin A + 80 ng/ml FGF2 + 3 µM CHIR99021 (CT99021; Stratech Scientific) + 10 µM LY (V1201; Promega) + 10 ng/ml BMP4 (R&D Systems), followed by day 2 media, consisting of CDM-PVA media + 100 ng/ml Activin A + 80 ng/ml FGF2 + 10 µM LY + 10 ng/ml BMP4, and day 3 media, consisting of RPMI + 2% B27 + 100 ng/ml ActiviNa + 80 ng/ml FGF2.
6
Differentiation of mESCs into Dopaminergic Neurons
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R1 mESCs (Nagy’s lab, MSH, Toronto, Canada) were cultured and differentiated on PA6 stromal feeders (RCB1127, Riken BRC Cell Bank, Japan)43 (link). Specifically, mESCs were plated at low density (100 cells/cm2) on a confluent layer of PA6 cells and were grown in Serum Replacement Medium with Noggin (300 ng/ml; R&D Systems) as previously described44 (link). At day 5, 200 ng/ml Shh (R&D Systems) and 25 ng/mL Fgf8b (R&D Systems) were added to the medium. At day 8, the medium was switched to N2 medium containing Shh, Fgf8b, and Fgf2 (10 ng/ml, R&D Systems). At day 10 of differentiation the cells were pulsed with EdU (10 μM, Life Technologies). From day 11 of differentiation, Shh, Fgf8b and Fgf2 were removed from the N2 medium and replaced by BDNF (20 ng/ml, R&D Systems), GDNF (20 ng/ml, R&D Systems), and ascorbic acid (0.2 mM). Between day 8–15, cells were treated daily with either dopamine (10 μM), haloperidol (1 μM), quinpirole (10 μM), sulpiride (10 μM) or media only. At day 15, cells were fixed in 4% PFA and processed for immunocytochemistry as described below.
7
Isolation and Activation of Muscle Stem Cells
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Muscle stem cells were isolated by FACS using a triple-negative CD31−/CD45−/Sca1− and double-positive VCAM+/α7-integrin+ strategy as described previously (Chakkalakal et al., 2012 (link)). Antibody clones, sources and catalog numbers are described in supplementary Materials and Methods . Cells were seeded in sarcoma-derived extracellular matrix (ECM)-coated 96-well plates in rich growth medium [F10 (Gibco), 20% fetal bovine serum (FBS) (Gibco), (5 ng/ml) FGF2 (R&D Systems)] for behavior analysis. For single cell sequencing experiments, cells were seeded in sarcoma-derived ECM-coated 6-well plates and allowed to activate in plating media [Dulbecco's modified eagle media (DMEM), 10% horse serum] for 18 h prior to library preparation.
Cells for behavior analysis were seeded at 850 cells/well on sarcoma-derived ECM (Sigma-Aldrich) in 96-well plates. Cells were maintained in rich growth media [F10 (Gibco), 20% FBS (Gibco), (5 ng/ml) FGF2 (R&D Systems)]. For single cell sequencing experiments in which cells were activated, cells were seeded in sarcoma-derived ECM-coated 6-well plates and allowed to activate in plating media (DMEM, 10% horse serum) for 18 h prior to library preparation.
Cells for behavior analysis were seeded at 850 cells/well on sarcoma-derived ECM (Sigma-Aldrich) in 96-well plates. Cells were maintained in rich growth media [F10 (Gibco), 20% FBS (Gibco), (5 ng/ml) FGF2 (R&D Systems)]. For single cell sequencing experiments in which cells were activated, cells were seeded in sarcoma-derived ECM-coated 6-well plates and allowed to activate in plating media (DMEM, 10% horse serum) for 18 h prior to library preparation.
8
Isolation of Primary Osteoblast Cells
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Primary osteoblast cells were isolated as previously reported by our group [21 (link)]. Briefly, crushed bones (femora, tibiae, and humeri) were removed from control or 5FU-treated mice and washed with PBS (GIBCO, Grand Island, NY) with 2% FBS (Hyclone, South Logan, UT) until the bone chips were white. Endosteal osteoblasts were released by enzyme digestion for 90 mins at 37 °C at 150 rpm with 3 mg/ml type I collagenase (Worthington, Lakewood, NJ) and 0.05% dispase (GIBCO) in PBS with 20% FBS. The released endosteal osteoblasts were washed in PBS + 2% FBS, and after removing the residual bone material osteoblasts were made into a single cell suspension by filtering through a 45-μm filter (BD Bioscience, San Jose, CA). In some experiments, osteoblastic cells were also isolated by the explant culture method in a 12-well plate containing the same weight of bone tissues per well. After primary osteoblasts became confluent within 5 to 10 days in the presence of FGF2 (R&D systems, Minneapolis, MN) or without FGF2, osteoblast colonies originating from bone fragments were washed with PBS (phosphate-buffered saline) and stained with a methanol-crystal violet solution (0.4%; wt/vol). Bones from non-5FU-treated mice were processed as a control for osteoblast growth recovery.
9
Cardiac Reprogramming of Induced Cardiomyocyte Progenitors
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For expansion, purified iCMPs were plated (5000/cm2) into gelatin-coated vessels and maintained in cardiac reprogramming medium with supplements (see below) for 27 days. Every 3–4 days, at ~ 80% confluency, iCMPs were sub-cultured (5000/cm2). Viable cell counts were determined using trypan blue (Sigma-Aldrich) and cumulative population doubling levels (PDLs) were calculated using the following formular: PDLharvested cells = 3.32[log(count cell harvest)–log(cell number plating)] + PDLplated cells. During expansion, cell morphology and Myh6/7 expression were assessed regularly by microscopy and flow cytometry (see above), respectively.
iCMP phenotype maintenance was examined in basic cardiac reprogramming medium or in the presence of various supplements: (1) geneticin (0.5 mg/ml); (2) ascorbic acid (250 µg/ml, FUJIFILM Wako Chemicals Europe, cat. no. 13–19,641); (3) FGF2 (2 ng/ml) and VEGFA (5 ng/ml) (both PeproTech); (4) FGF2 (2 ng/ml), VEGFA (5 ng/ml), and BMP4 (20 ng/ml; R & D Systems); and (5) FGF2 (2 ng/ml) and BMP4 (20 ng/ml) [27 (link)]. Media were changed every other day.
iCMP phenotype maintenance was examined in basic cardiac reprogramming medium or in the presence of various supplements: (1) geneticin (0.5 mg/ml); (2) ascorbic acid (250 µg/ml, FUJIFILM Wako Chemicals Europe, cat. no. 13–19,641); (3) FGF2 (2 ng/ml) and VEGFA (5 ng/ml) (both PeproTech); (4) FGF2 (2 ng/ml), VEGFA (5 ng/ml), and BMP4 (20 ng/ml; R & D Systems); and (5) FGF2 (2 ng/ml) and BMP4 (20 ng/ml) [27 (link)]. Media were changed every other day.
10
Differentiation of hPSCs into Hepatocytes
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hPSCs were differentiated as discussed previously (Si-Tayeb et al. 2010 (link); Mallanna and Duncan 2013 ; Yanagihara et al. 2016 (link)). Briefly, hPSCs were harvested using Accutase and plated at 600,000 cells per well in 24-well plates precoated with 300 µl/well Geltrex (Thermo Fisher Scientific, Waltham, MA). Approximately 24 h after seeding the cells with mTeSR1 (Stemcell Technologies), when the cells were 85–95% confluent, differentiation was initiated by culture for 5 d with 50 ng/ml Activin A (R&D Systems) in RPMI/B27 (without insulin) supplement (Invitrogen) under ambient oxygen/5% CO2. In addition, we included 10 ng/ml BMP4 (R&D Systems) and 20 ng/ml FGF2 (R&D Systems) for the first 2 d. Then, the cells were cultured for 5 d with 20 ng/ml BMP4 (R&D Systems)/10 ng/ml FGF-2 (R&D Systems) in RPMI/B27 (containing insulin) under 4% O2/5% CO2, then for 5 d with 20 ng/ml HGF (R&D Systems) in RPMI/B27 (containing insulin) under 4% O2/5% CO2, and finally for 5 d with 20 ng/ml Oncostatin-M (R&D Systems) in Hepatocyte Culture Media (Lonza) supplemented with SingleQuots (without EGF) in ambient oxygen/5% CO2.
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