Optimized DNA sequence encoding Human TNF beta mature chain was expressed in Escherichia Coli
Native human Tumor Necrosis Factor-beta is generated by the proteolytic removal of the signal peptide and propeptide, this molecule has a calculated mass of approximately 19 kDa. Recombinant TNF-beta is a non disulfide-linked homotrimer protein,each consisting of 172 amino acids and migrates as an approximately 19 kDa protein under reducing conditions.
>95%, as determined by SDS-PAGE and HPLC
The ED(50) was Measured in a cytotoxicity assay using mouse L929 cells in the presence of the metabolic inhibitor actinomycin, and was determined to be less than.1ng/ml.
MTPPERLFLP RVCGTTLHLL LLGLLLVLLP GAQGLPGVGL TPSAAQTARQ HPKMHLAHST LKPAAHLIGD PSKQNSLLWR ANTDRAFLQD GFSLSNNSLL VPTSGIYFVY SQVVFSGKAY SPKATSSPLY LAHEVQLFSS QYPFHVPLLS SQKMVYPGLQ EPWLHSMYHG AAFQLTQGDQ LSTHTDGIPH LVLSPSTVFF GAFAL
Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than.1 ng/µg(1EU/µg).
TNF-beta is presented as a.2 μm filtered PBS solution pH7.5.
A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than.1 mg/mL. This solution can then be diluted into other buffers
The lyophilized protein is stable for at least years from date of receipt at -20° C. Upon reconstitution, this cytokine can be stored in working aliquots at° -° C for one month, or at -20° C for six months, with a carrier protein without detectable loss of activity. Avoid repeated freeze/thaw cycles.
This cytokine product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.
Release of sST2 from placenta explants
- All placentas were from pregnant, healthy women undergoing elective caesarean section, without labor, and were processed immediately.
- Freshly delivered placentae (n = 3) were first rinsed in ice cold Hanks balanced salt solution and placed into a glove box maintained at 8% O2.
- Placental pieces, cut from undamaged lobules that appeared healthy, were rinsed in 8% O2 equilibrated ice cold explant culture medium'>medium medium'>(DMEM containing 10% foetal bovine serum , 1% antibiotic and antimycotic solution and L-glutamine) before being placed into ice cold fresh equilibrated explant culture medium'>medium.
- Placental pieces of approximately 2 mm in diameter were then dissected and distributed equally between Costar Netwell (24 mm diameter, 500 µm mesh) supports in 6-well plates containing 4 ml/well equilibrated explant culture medium (10 explants/well).
- Placental explants were finally washed again with a medium change before being incubated under conditions ‘normoxic’ for trophoblast (8% O2/87% N2/5% CO2)…
- Recombinant human TNF-α was from .
- Myosin Light Chain Kinase inhibitor PIK was generated as described
- After 2 passages, cells were seeded into 6-well plates .
- At confluence, cytokine treatment was performed during 48 hours using human recombinant TNF-α 100 U/ml or IFN-γ 200 U/ml in serum-free media, otherwise as above.
- Experiments were ended by removal and freezing of the supernatants and addition of lysis buffer to the cell monolayer, see below.
MSCs and SB623 cells interfere with monocyte-DC differentiation and maturation.
- Representative FACS data (above) and mean expression for 3 different matched lots of MSC and SB623 cells (below); the percentage of cells co-expressing CD14 and CD1A after GM-CSF+IL-4 culture is significantly higher when monocytes were cultured with either MSCs or SB623 cells versus alone (#p < 0.05); the percentage of CD14-CD1A+ cells is significantly lower when differentiation culture included either MSCs or SB623 cells versus alone (*p < 0.05); C) CD86 surface expression after 2-day TNF-α treatment; Representative FACS data (left) and mean fluorescence intensity for 3 different matched lots of MSC and SB623 cells (right); there is a significant decrease in the mean fluorescent intensity of CD86 co-stimulatory molecule when monocytes were co-cultured with SB623 cells versus parental MSCs (*p < 0.05).
A: cumulative plot showing the time from start of the experiment, at which neurons with baseline ongoing activity were identified in the untreated group with peripheral field searches (paradigm 2; n = 30 neurons) and the neuritis group (paradigm 1; n = 36 neurons).
- B: percentage of neurons (silent and ongoing) recorded early (<151-min postsetup) and late (>151 min) in the untreated group with peripheral field searches (paradigm 2) that responded to cytokine treatment (CCL2 and TNF-α combined).
ELISA for TNFα and IL6
- Concentrations of TNF-α in supernatants were determined using the ELISA development kit supplied by .
Proliferation, collagen expression, and basal MMP expression of cardiac, dermal, and pulmonary fibroblasts.
- (b) Collagen mRNA expression level was determined using TaqMan from fibroblasts incubated with or without 10 ng/mL TNF-α for 24 hours.
γδ T-cells within BCG-, M. vaccae- and M. obuense-treated PBMCs produce granzyme B and TH1 cytokines.
- e Percentages of γδ T-cells expressing IFN-γ, TNF-α and IL-10 were measured after 24 h of stimulation.
Ectopic expression of Id1 potentiates NF-κB signaling in kidney tubular epithelial cells (a, b) Id1 potentiates IκBα phosphorylation after TNF-α stimulation.
- Id1-overexpressing cells and pcDNA3-mock transfection controls were treated with TNF-α (5 ng/ml) for various periods of time.
Circumferential podosomes/invadopodia mediate invasive and fusion activities.
- B16F0 murine melanoma cells transfected with control (Ctr) or Tks5 siRNAs were cultured in the presence of 10 ng/ml RANKL alone or together with 5 ng/ml TGF-β and 5 ng/ml TNF-α for 48 h. (left) The cells were then stained with rhodamine-phalloidin to visualize F-actin, with antibodies to Tks5, and with DAPI to visualize nuclei.