Certificate of Analysis Vitronectin, Human: Description: Human Vitronectin is a major plasma glycoprotein that exhibits multiple activities and functions as a cell adhesion molecule and regulator of coagulation. It contains the amino acid structural motif, Arg-Gly-Asp (RGD), which is involved in cell attachment. Human Vitronectin has a mass of 75kDa and circulates as a single-chain moiety of 75kDa and a two-chain moiety of 65kDa and 10kDa. Human Vitronectin is purified from human plasma.
Vitronectin belongs to the group of structurally and functionally homologous adhesive proteins (fibrinogen, fibronectin, Von Willebrand factor), which interact with platelets and the vessel wall in the early stages of blood clotting. When coated onsurfaces, very low concentrations of Vitronectin promote endothelial cell attachment and induce spreading and migration ofcells in a time- and concentration-dependent fashion (1). Concentration: 2mg/ml. Source: Human Vitronectin is purified from human plasma. Storage Buffer: Human Vitronectin is supplied in Dulbecco’s PBS (calcium- and magnesium-free) containing 175mM ammonium sulfate. Storage Conditions: See the product information label for storage recommendations. Avoid multiple freeze-thaw cycles and exposure to frequent temperature changes. These fluctuations can greatly alter product stability. Human Vitronectin can be further diluted in DPBS but should not be stored as a dilute solution. See the expiration date on the product information label. Promega Corporation Quality Control Assays
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Biological Activity: When coated onto tissue culture plastic, Human Vitronectin promotes one half maximal attachment of
BALB/3T3 cells in serum-free medium at <0.1µg/cm2. Maximum attachment should occur at approximately 0.2µg/cm2. Virus Contamination: Human Vitronectin is purified from human plasma that has been tested by the American Red Cross
and found to be nonreactive for HIV-1 and Human T-cell leukemia virus type I (HTLV-I) antibody and antigen, Hepatitis Bsurface antigen (HBsAg) and antibodies to Hepatitis C. These results are obtained using standard serologic tests for detec-tion of antibodies and viral antigens in human blood and tissues. Caution: These results are subject to the limitations of the
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virus detection assays. No known test methods can offer assurance that products derived from human tissue will not transmit
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infectious agents. Observe universal precautions when working with this product.
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CellTiter 96 is a registered trademark of PromegaCorporation.
Products may be covered by pending or issuedpatents or may have certain limitations. Please visitour Web site for more information.
All specifications are subject to change without priornotice.
Product claims are subject to change. Please contactPromega Technical Services or access the Promegaonline catalog for the most up-to-date information onPromega products. Usage Information I. General Protocol for Coating Substrates with Attachment
Place the flask of cells from the 37°C incubator directly on the bag of crushed ice,
allow to cool for 3–4 minutes, and then remove the medium.
Rinse the flask with sterile, ice-cold DPBS. Materials to Be Supplied by the User
Remove the DPBS and add 2ml of ice-cold 0.2µm filter-sterilized 0.05% (w/v) trypsin
(Solution composition is provided in Section III.)
dissolved in DPBS. Note: Store stock solutions of trypsin at –20°C in 2ml aliquots.
Thaw immediately before use and keep on ice.
2mg/ml sterile bovine serum albumin (BSA) in DPBS
After 1 minute, aspirate the trypsin solution and let the plate stand on the ice bag for
an additional 3 minutes or until the cells round up and begin to detach when the flask
Dilute the stock solution to the desired concentration with DPBS to obtain the final
coating solution. To coat plastic surfaces, add 100µl of the various concentrations of
Gently remove the monolayer of cells from the plastic surface by using a pipette to
attachment factor to replicate sets of a 96-well tissue culture plate. Use 0–1µg/cm2
add 5ml of ice-cold 0.2µm filter-sterilized 0.2% (w/v) soybean trypsin inhibitor
dissolved in DPBS. Note: Store aliquots of soybean trypsin inhibitor at –20°C and
Coat substrates with enough final coating solution to completely cover the surface.
Incubate for 2 hours at room temperature.
Transfer the solution to a 15ml sterile conical polypropylene centrifuge tube and
Remove the coating solution and immediately rinse the wells twice with DPBS.
bring the volume up to 10ml by adding SFM.
Add 200µl/well of sterile 2mg/ml BSA in DPBS.
Centrifuge at 300 × g for 4 minutes at 4°C.
Incubate the plate for 1 hour at 37°C.
Aspirate the supernatant, gently resuspend the cell pellet in 10ml of ice-cold SFM
Remove the BSA solution before adding cells. II. Sample Protocol for Determining the Optimal
10. Aspirate the supernatant and gently resuspend the cell pellet in 10ml ice-cold SFM. Concentration of Human Vitronectin to Facilitate
11. Using a hemocytometer, determine cell number and viability by counting trypan
Attachment of BALB/3T3 Cells Materials to Be Supplied by the User III. Composition of Buffers and Solutions
(Solution compositions are provided in Section III.)
Ham's F12/Dulbecco's modified Eagle’s medium (F12/DME)
0.05% (w/v) trypsin in DPBS (0.2µm filter-sterilized)
0.2% (w/v) soybean trypsin inhibitor (0.2µm filter-sterilized)
CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Cat.# G5421)
Three to four days prior to the assay, seed a 75cm2 flask with BALB/3T3 clone A31(ATCC CCL 163) cells at 1–2 × 105 cells in F12/DME with 10% calf serum.
Add 1:1 v:v ratio of Ham's F12 and Dulbecco's modified Eagle’s medium. Supplement to
Harvest the cells using ice-cold trypsinization procedure (see Section II.B).
Resuspend the harvested cells at 3.5 × 105 cells/ml in serum-free medium.
Add 100µl (3.5 × 104 cells) to each well that has been coated with varying concentrations of Vitronectin (see Section I.A, Step 1).
Incubate plates at 37°C in a humidified, 5% CO
Supplement the F12/DME to contain a final concentration of:
After 1 hour, gently remove the medium and unattached cells with a multichannel
Using a multichannel pipette, wash the wells three times by addition and subsequent
removal of 200µl/well of F12/DME at 37°C.
Add 20µl of freshly prepared MTS/PMS solution CellTiter 96® AQ
IV. Related Products
10. Incubate the plate at 37°C in a humidified, 5% CO2 atmosphere for 1–4 hours.
CellTiter 96® AQueous Non-Radioactive 1,000
11. Record the absorbance at 490nm using an ELISA plate reader.
12. Plot the corrected absorbance at 490nm (Y axis) versus concentration of Vitronectin
50 value, find the X-axis value that corresponds to one
half the difference between the maximum (plateau) and minimum (no Vitronectin
V. References B. Ice-Cold Trypsinization Procedure (2)
1. Preissner, K.E. (1989) The role of vitronectin as multifunctional regulator in the
hemostatic and immune systems. Blut 59, 419–31.
Place crushed ice in a sealable plastic bag. Remove all the air from the bag by adding
2. Riss, T.L. et al. (1988) Human recombinant insulin-like growth factor I.
water or letting the ice melt to a slush consistency. Wipe the bag with 70% ethanol
I. Development of a serum-free medium for clonal density assay of growth factors
and place the bag in a laminar flow hood.
using BALB/c 3T3 mouse embryo fibroblasts. In Vitro Cell. Dev. Biol. 24, 1099–106.
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C U R R I C U L U M V I T A E FULL NAME : DR. ALFRED M.E. NABURI Tel/Fax ++255-27-2753702/2750330 RDTC. DATE OF BIRTH 26th June, 1958 PLACE OF BIRTH Koboko North Village, Nasai Ward, Livishi Division, Siha District, Kilimanjaro region, Tanzania. NATIONALITY Tanzanian MARITAL STATUS Married with two children QUALIFICATIONS - Master of Public Health - KCM College of the Tumain