Bovine ECGFpro1 (complete for blood ECs) Protein

Specifications

Product Data
Description	Bovine ECGFpro1 (complete for blood ECs) recombinant protein, 6 mg
Protein Source	Brain
Purity	Crude extract.
Buffer	State: Sterile, lyophilized, Freeze dried powder Buffer System: H2O without preservative
Reconstitution	To obtain a stock solution reconstitute the contents of the vial in 2 ml of prewarmed (37 °C) sterile PBS or water. Gently rotate the vial until the contents are dissolved. This stock solution may be further diluted in sterile tissue culture media to obtain the desired working concentrations. Although the stock solution can be added aseptically to sterile tissue culture medium, it is recommended that medium containing diluted product is aseptically filtered prior to use. The ECGF + VEGF-A are sufficient for 500 ml growth medium.
Preparation	Sterile, lyophilized, Freeze dried powder
Applications	Biological activity/ Working concentration: Optimum concentration for human umbilical vein endothelial cells (HUVEC) range from 50-200 μg/ml, optimal concentration with heparin (50 μg/ml) is about 12 μg/ml. Protocol: Thymidine Incorporation with HUVEC - plate cells with a density at 5-7 x 10e3 cells/well in growth medium (EGM) - incubate cells over night [if urgent, plate cells in the morning, change growth medium against basal medium (EBM) in the early afternoon] - change EGF against EBM (for HUVEC: EBM +1-2% FCS) - incubate 24 h - change medium again and add factors (growth factors, inhibitors, etc.) - incubate for 18 h - add 10 µl 3H-Thymidine solution [0.025 mCi/ml] per well (=0.25 µCi) - incubate another 6h at 37°C - Washing steps: (250 µl/well) PBS 1x MeOH 2x 5 min TCA 2x 10 min H2O 1x - lyse cells in 250 µl 0.3M NaOH per well - transfer 2.5 ml ECO Plus into the appropriate scintillation vials - transfer cell lysats into the scintillation vials - count by liquid scintillation (ß-counter; Beckmann Instruments)
Protein Description	Endothelial cell growth factor (ECGF) is an extract of bovine brain containing growth promoting factors for vascular endothelial cells of mammalian origin. ECGF has also been reported to be beneficial as a media supplement for the fusion and growth of hybridoma cells in monoclonal antibody production. Endothelial cell growth factor is prepared using a modification of the method of Maciag, et al. (1979) lyophilized from a sterile solution containing NaCl and streptomycin sulfate. ECGFpro1 is supplemented with recombinant human VEGF165 (corresponding to 10 ng/ml) a concentration sufficient for the cultivation of blood vascular endothelial cells like HUVEC and HMVEC. Species specificity: Bovine ECGF is effective on Mouse, Bovine and Human cells.
Note	Additional Factor: rh-VEGF165 (final concentration: 10ng/ml)
Storage	Store lyophilized at 2-8°C for 6 months or at -20°C long term. After reconstitution store the antibody undiluted at 2-8°C for one month or (in aliquots) at -20°C long term. Avoid repeated freezing and thawing.
Stability	Shelf life: one year from despatch.
Reference Data
Summary	Endothelial cell growth factor (ECGF) is an extract of bovine brain containing growth promoting factors for vascular endothelial cells of mammalian origin. Endothelial cell growth factor is prepared using a modification of the method of Maciag, et al. (1979) lyophilized from a sterile solution containing NaCl and streptomycin sulfate. ECGFpro1 is supplemented with recombinant human VEGF165 (corresponding to 10 ng/ml) a concentration sufficient for the cultivation of blood vascular endothelial cells like HUVEC and HMVEC. Endothelial cells from human umbilical vein (HUVEC) can be established as primary cultures by traditional methods. The serial propagation of these cells has proved to be difficult. The long-term propagation of these cells in vitro can be achieved with an extract prepared from bovine brain. The introduction of a fibronectin or collagen matrix to the cell culture system allows cultivating endothelial cells at clonal densities. With ECGF, the FCS requirement can be reduced. Heparin potentiates the mitogenic activity of crude preparations of ECGF. ECGF has also been reported to eliminate the need for feeder cells in the clonal growth of hybridomas and other cell types.
Protein Families	Biological activity/ Working concentration: Optimum concentration for human umbilical vein endothelial cells (HUVEC) range from 50-200 μg/ml, optimal concentration with heparin (50 μg/ml) is about 12 μg/ml. Protocol: Thymidine Incorporation with HUVEC - plate cells with a density at 5-7 x 10e3 cells/well in growth medium (EGM) - incubate cells over night [if urgent, plate cells in the morning, change growth medium against basal medium (EBM) in the early afternoon] - change EGF against EBM (for HUVEC: EBM +1-2% FCS) - incubate 24 h - change medium again and add factors (growth factors, inhibitors, etc.) - incubate for 18 h - add 10 µl 3H-Thymidine solution [0.025 mCi/ml] per well (=0.25 µCi) - incubate another 6h at 37°C - Washing steps: (250 µl/well) PBS 1x MeOH 2x 5 min TCA 2x 10 min H2O 1x - lyse cells in 250 µl 0.3M NaOH per well - transfer 2.5 ml ECO Plus into the appropriate scintillation vials - transfer cell lysats into the scintillation vials - count by liquid scintillation (ß-counter; Beckmann Instruments)

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