OVERVIEW
Here we describe a protocol to coat glass coverslips with poly-d-lysine. This protocol coats the coverslips uniformly, is rapid and consumes very little amount of poly-d-lysine. However, it coats only one side of the coverslip which is sufficient for most applications. If you want to coat both sides, you must repeat the procedure with the other side.
In this protocol, a 22 mm coverslip is placed on a 50 – 60 µl drop of 0.1 mg/ml poly-d-lysine solution and is incubated for 30 min at 37°C in a humidified atmosphere (e.g., cell culture incubator). After incubation, the coverslip is rinsed with Milli-Q water, dried, and stored at 4°C. Coated coverslips can be used to seed mammalian cells.
REQUIREMENTS
Reagents and solutions
♦ 0.1 mg/ml poly-d-lysine solution (sterile)
♦ MiliQ water (Sterile)
Equipment and disposables
♦ Petri dish
♦ Parafilm
♦ 37°C incubator *
♦ Micropipette and micropipette tips
♦ Laminar flow hood (optional)
* Cell culture incubator can be used
OBJECTIVE
Coating glass coverslips with 0.1 mg/ml Poly-D-Lysine
PROTOCOL:
Use appropriate personal protective equipment (lab coat, gloves, goggles, etc) for your safety and follow your institute’s guidelines.
Use sterile coverslips and do all steps in sterile conditions (use a Laminar flow hood) if you want coated coverslips to be sterile!
Step 1: Place a right-size parafilm in a Petri dish (e.g., 10 cm), and press it gently so that it is fixed to the Petri dish.
Tips:
1. Depending on the number of coverslips, you can choose the petri dish and parafilm size. A 10 cm Petri dish is sufficient to coat 4 coverslips.
2. Parafilm should be flat without any ridges.
How to maintain sterile conditions
- Clean the parafilm with 75% ethanol and dry it inside the laminar flow hood.
- Use sterile Petri dishes.
- Fix the parafilm in petridish and UV-irridiate (can be done in laminar flow hood) for 20 min.
Step 2: Take 50 – 60 µl of 0.1 mg/ml poly-d-lysine solution and place it on parafilm. The solution will form a drop on parafilm. Take a 22 mm coverslip and put it on the drop of parafilm solution.
Tips: For an 18 mm coverslip, 40 – 50 µl of 0.1 mg/ml poly-d-lysine solution is sufficient
Precaution: There will be a thin layer of liquid between the coverslip and parafilm. Make sure that solution covers the entire surface of the coverslip and there should not be any air bubbles trapped between the parafilm and the coverslip.
Step 3: Cover the Petri dish with the lid and place it in a humidified 37°C incubator for 30 min (e.g., cell culture incubator).
Tips:
1. If a humidified incubator is not available, you can place some wet tissue papers in the Petri dish. A humid environment will reduce the evaporation of water from the poly-d-lysine solution.
2. Repeat steps 2 and 3 with the other side if you want to coat both sides of the coverslip.
Step 4: After incubation, take the coverslip and rinse it with Mili Q water once and dry.
Tips: To keep the coverslip sterile, you must do this step under sterile conditions (inside the laminar flow hood) and use autoclaved MiliQ water.
Step 6 (optional): You can sterilize the coverslip by keeping it in UV light for 30 min.
We recommend doing this step if you want to use coated coverslips for cell culture work. You can keep the dried coverslip in the laminar flow hood and turn the UV on for 30 min.
STORAGE
Poly-D-lysine-coated coverslips can be stored at 4°C for a month.
