Coating Tissue Culture Plates with Vitronectin

Product No. SRP3186

Vitronectin is used to coat culture vessels for cells attachment. Below is a suggested coating protocol for a 96-well plate, at a coating concentrations of 0.5 µg/cm2. Note that the optimal concentration vary with each cell line. For culture vessels other than 96-well, this protocol should be scaled according to Table 1. To calculate the working concentration of vitronectin use Table 1, or use the following equation:

Working concentration calculation Example:

To coat a 96-well plate (surface area is 0.3 cm2/well) at a coating concentration of 0.5 µg/cm2, prepare 9.6-10 ml of diluted vitronectin solution (0.1 ml/well × 96 wells, see Table 1) at the following concentration:

The vitronectin working concentration in this example is 1.5 µg/ml. Adding 0.1 ml per well of this working solution will yield a coating concentration of 0.5 µg/cm2.

 

Bioassay
The biological activity of human, recombinant Vitronectin (VTN) expressed in HEK 293 cells (Cat.No. SRP3186) was measured in a cell attachment assay using CHO cells. The EC50 is defined as the effective concentration that elicits a 50% increase in cell attachment in a cell based bioassay.

Purity
The purity of human, recombinant Vitronectin expressed in HEK 293 cells (Cat. No. SRP3186) was assessed on SDS-PAGE (1.25 μg/lane.) followed by InstantBlue™ staining (Cat. No. ISB1L). (Representative result

Materials

  1. 1X PBS
  2. Pure water (e.g., Cat. No W3500)
  3. Sterile tissue culture 96-well plate with a lid
  4. Recombinant human vitronectin (Cat. No. SRP3186)

Protocol

A. Preparation of vitronectin

  1. Reconstitute the lyophilized vitronectin with sterile water, to a final concentration of 100-300 µg/ml. This stock solution can be stored at 2-8 oC for up to one week. For extended storage, it is recommended to further dilute in a buffer containing a carrier protein (example 0.1% BSA) and store in working aliquots at –20 oC to –80 oC.
  2. Further dilute the reconstituted vitronectin stock solution in 1X PBS to a final concentration of 1.5 µg/ml. For example, for a 10 ml solution (sufficient to coat one 96-well plate), dilute 75 µl of 200 µg/ml stock solution in 10 ml of 1X PBS.

B. Coating the plate

  1. To each well, add 100 µl of the 1.5 µg/ml vitronectin solution, for a final coating concentration of 0.5 µg/cm2.
  2. Cover the plate with a lid. For best results it is recommended to incubate the plate for 2 hours at 37 oC in 5% CO2 and 95% air, and then over-night at 2-8 oC.
  3. Aspirate the vitronectin solution, being careful not to scratch the coated vitronectin. The plate is now ready to use.
  4. Wash each well 3 times with 1XPBS.
  5. For immediate use, aspirate the 1XPBS. Alternatively, the coated plate can be stored for up to one week at 2-8 oC with 1XPBS, covered and wrapped in laboratory film.
  6. Prior to use, pre-warm the coated plate.

Table 1: Volumes and working concentrations of vitronectin for various tissue culture vessels
 

Culture Dish Approx. surface area Diluted vitronectin volume Vitronectin working concentration
      for 0.1 µg/cm2 for 0.5 µg/cm2 for 1 µg/cm2
96-well plate 0.3 cm2 0.1 ml/well 0.3 µg/ml 1.5 µg/ml 3 µg/ml
24-well plate 2 cm2 0.2 ml/well 1 µg/ml 5 µg/ml 10 µg/ml
12-well plate 4 cm2 0.4 ml/well 1  µg/ml 5 µg/ml 10 µg/ml
6-well plate 9 cm2 1 ml/well 0.9  µg/ml 4.5 µg/ml 9 µg/ml
35 mm plate 9 cm2 1 ml 0.9  µg/ml 4.5 µg/ml 9 µg/ml
60 mm plate 21 cm2 1 ml 2.1 µg/ml 10.5 µg/ml 21 µg/ml
100 mm plate 55 cm2 2 ml 2.75  µg/ml 13.75 µg/ml 27.5 µg/ml
150 mm plate 152 cm2 6 ml 2.53 µg/ml 12.67 µg/ml 25.33 µg/ml
T-25 flask 25 cm2 2.5 ml 1 µg/ml 5 µg/ml 10 µg/ml
T-75 flask 75 cm2 7.5 ml 1 µg/ml 5 µg/ml 10 µg/ml