Coating protocols

Protocol for poly-lysine coating a microcell array

Materials

Either poly-lysine or poly-D-lysine with MW between 30,000 - 150,000 can be used for the coating.

Prepare 1 mg/mL stock solution by dissolving 100 mg of poly-D-lysine hydrobromide in 100 mL of PBS. The stock solution can be stored at -20°C.

The working solution is obtained by diluting the stock solution with PBS to around 0.1 mg/mL.

Procedure

1.

Insert the microcell array into the cell culture ware to be used for imaging.

2.

Using a 1,000 μL pipette slowly add 500 μL of 100% ethanol on top of the microcell array, then cycle the ethanol over the microcell array to assist in removing any trapped air bubbles.

3.

Slowly add media to one corner of the cell culture ware until almost full.

4.

Draw out the media/ethanol mixture from the opposite corner of the culture ware, taking care that the microcell array is always covered with a little amount of liquid. Repeat steps 2 and 3 at least five times to make sure that the ethanol is completely diluted out.

5.

Add the working solution of poly-lysine to the microcell array.

6.

Incubate for 1 hour at room temperature in a hood.

7.

Carefully aspirate the poly-lysine solution from the culture ware, taking care that the microcell array is always covered with a little amount of liquid. Note that the poly-lysine working solution can be re-used a few times.

8.

Rinse 3 times with PBS, taking care that the microcell array is always covered with a little amount of liquid.

9.

The poly-lysine coated microcell array should be used immediately.

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Preparation of poly-lysine, fibronectin, collagen and laminen surface coatings for microcell, microgrid, micromesh and microwell arrays

Poly-lysine, fibronectin, collagen and laminen surface coatings for microcell, microgrid, micromesh and microwell arrays

Rinsing poly-lysine, fibronectin, collagen and laminen surface coatings for microcell, microgrid, micromesh and microwell arrays
Protocol for collagen coating a microcell array

Materials

Rat tail collagen (type 1) can be used for the coating. It is recommended that you purchase collagen solutions.

Dilute the purchased solution to 50 μg/mL with 0.02M acetic acid to produce the working solution.

Procedure

1.

Insert the microcell array into the cell culture ware to be used for imaging.

2.

Using a 1,000 μL pipette slowly add 500 μL of 100% ethanol on top of the microcell array, then cycle the ethanol over the microcell array to assist in removing any trapped air bubbles.

3.

Slowly add media to one corner of the cell culture ware until almost full.

4.

Draw out the media/ethanol mixture from the opposite corner of the culture ware, taking care that the microcell array is always covered with a little amount of liquid. Repeat steps 2 and 3 at least five times to make sure that the ethanol is completely diluted out.

5.

Add the working solution of collagen (50 μg/mL) to the microcell array at 5 μg/cm2.

6.

Incubate for 1 hour at room temperature in a hood.

7.

Carefully aspirate the collagen solution from the culture ware, taking care that the microcell array is always covered with a little amount of liquid. Alternatively the culture ware with the microcell array can be left in the hood to dry overnight and the following wash step is not needed.

8.

Rinse 3 times with PBS or serum-free media, taking care that the microcell array is always covered with a little amount of liquid.

9.

The collagen coated microcell array should be used immediately.

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Protocol for fibronectin coating a microcell array

Materials

Human fibronectin is diluted to 10 - 50 μg/mL with Ca++ and Mg++ free PBS to produce the working solution. Note that the concentration should be proportional to the coating concentration. For example, make 10 μg/mL solution for 1 μg/cm2 coating and 50 μg/mL solution for 5 μg/cm2 coating.

Make appropriate aliqouts and store at -20°C and avoid multiple freeze/thaw cycles.

Procedure

1.

Insert the microcell array into the cell culture ware to be used for imaging.

2.

Using a 1,000 μL pipette slowly add 500 μL of 100% ethanol on top of the microcell array, then cycle the ethanol over the microcell array to assist in removing any trapped air bubbles.

3.

Slowly add media to one corner of the cell culture ware until almost full.

4.

Draw out the media/ethanol mixture from the opposite corner of the culture ware, taking care that the microcell array is always covered with a little amount of liquid. Repeat steps 2 and 3 at least five times to make sure that the ethanol is completely diluted out.

5.

Add the appropriate amount of fibronectin solution to the microcell array.

6.

Incubate for 1 hour at room temperature in a hood.

7.

Carefully aspirate the remaining solution from the culture ware, taking care that the microcell array is always covered with a little amount of liquid.

8.

Rinse 3 times with PBS, taking care that the microcell array is always covered with a little amount of liquid.

9.

The fibronectin coated microcell array should be used immediately.

Download the microcell array protocols
Protocol for laminin coating a microcell array

Materials

Laminin is diluted to 5 μg/mL in PBS. The concentration should be proportional to the coating concentration. For example, make 10 μg/mL solution for 1 μg/cm2 coating and 50 μg/mL solution for 5 μg/cm2 coating.

Make appropriate aliqouts and store at -20°C and avoid multiple freeze/thaw cycles.

Procedure

1.

Insert the microcell array into the cell culture ware to be used for imaging.

2.

Using a 1,000 μL pipette slowly add 500 μL of 100% ethanol on top of the microcell array, then cycle the ethanol over the microcell array to assist in removing any trapped air bubbles.

3.

Slowly add media to one corner of the cell culture ware until almost full.

4.

Draw out the media/ethanol mixture from the opposite corner of the culture ware, taking care that the microcell array is always covered with a little amount of liquid. Repeat steps 2 and 3 at least five times to make sure that the ethanol is completely diluted out.

5.

Add an appropriate amount of laminin solution to the microcell array.

6.

Incubate for 1 hour at room temperature in a hood.

7.

Carefully aspirate the remaining solution from the culture ware, taking care that the microcell array is always covered with a little amount of liquid.

8.

Rinse 3 times with PBS, taking care that the microcell array is always covered with a little amount of liquid.

9.

The laminin coated microcell array should be used immediately.

Download the microcell array protocols