Coating Tissue Culture Plates with Vitronectin

Vitronectin (Product No. SRP3186) is used to coat culture vessels for cells attachment. Below is a suggested coating protocol for a 96-well plate at a coating concentration of 0.5 µg/cm². Note that the optimal concentration will vary with each cell line. For culture vessels other than a 96-well plate, scale this protocol accordingly. To calculate the working concentration of vitronectin, use Table 1, or 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, 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 (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 (SRP3186) was assessed on SDS-PAGE (1.25 μg/lane.) followed by InstantBlue™ stain (ISB1L). (Representative result)

Materials

• 1X PBS
• Pure water (e.g.,Product No. W3500)
• Sterile tissue culture 96-well plate with a lid (Product No. CLS3300)
• Recombinant human vitronectin (Product 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 best to further dilute in a buffer containing a carrier protein (example 0.1% BSA) and store in working aliquots at -20 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 best 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. Before 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