Overview: How to coat Petri dishes with collagen

All three protocol steps involving liquid transfer were analyzed by measuring the time taken to process 100 Petri dishes, using either a pipette controller in combination with 10 ml serological pipettes or the DOSE IT peristaltic pump.

Before coating the Petri dishes, a collagen stock solution should be prepared according to the manufacturer’s instructions. This stock solution should be diluted to a final concentration of 50 μg/ml collagen in 0.02 M acetic acid.

Overview of the steps:

1. DOSE IT set-up
2. Coating the Petri dishes with collagen
3. Removing the excess collagen solution
4. Washing the coated Petri dishes

Step-by-step procedure:

1. DOSE IT set-up

Tubing adjustment, priming and calibration for dispensing the solution

Open the pump head, adjust the tubing clamps and insert sterilized 4.0 mm tubing into the DOSE IT. Set the flow rate to 470 ml/min (280.7 rpm) for optimal performance. Put the aspiration tube with the tube collar into the collagen solution, and the dispensing tube into an empty bottle to fill the empty tubing with liquid.

Perform the priming and calibration steps according to the Operating instructions.

2. Coating the Petri dishes with collagen

Dispensing the collagen solution into the Petri dish

Put the dispensing tube above the Petri dish. The DOSE IT directly dispenses the required volume of collagen solution into the Petri dish (Table 1). Make sure the entire bottom of the Petri dish is covered with collagen solution. After filling all the Petri dishes, remove the aspiration tube from the collagen solution and put the dispensing tube into an empty bottle. Incubate the Petri dishes at room temperature for one hour. The DOSE IT can also be used for other culture flasks or dishes, with dispensing volumes given in Table 1.

Tip:

• Optimizing collagen concentration and coating times for your cell lines and experimental needs is recommended.

Table 1: Typical coating and washing volumes.

4. Washing the coated Petri dishes

Rinsing the Petri dishes with PBS buffer to remove the remaining acid

Put the aspiration tube into the PBS buffer (or serum-free medium), and the dispensing tube into an empty bottle to refill the empty tubing. Fill the tubing with PBS buffer, then rinse the Petri dish twice with PBS to remove the remaining acid (see Table 1 for volumes). Air dry the Petri dishes at room temperature inside a laminar flow hood, then seal with parafilm and store at 4 °C until needed.

Results

For all steps, using the DOSE IT was significantly faster than working with a serological pipette controller, with an overall time saving of 40 % (Table 2).

Table 2: Measured time for handling 100 Petri dishes using a serological pipette controller or a DOSE IT peristaltic pump. Righthand column shows % time saving using a peristaltic pump.