Vitronectin. human, product information, 9pig538

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
Promega Corporation
Quality Control Assays
2800 Woods Hollow RoadMadison, WI 53711-5399 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 PRODUCT USE LIMITATIONS, WARRANTY, DISCLAIMER
virus detection assays. No known test methods can offer assurance that products derived from human tissue will not transmit Promega manufactures products for a number of infectious agents. Observe universal precautions when working with this product.
intended uses. Please refer to the product label forthe intended use statements for specific products.
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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 A. Protocol
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) A. Protocol
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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