Polylysine-coated tissue culture surfaces

Contents

  1. Introduction
  2. Preparation
  3. Coating culture dishes, multiwell plates, or chamber slides
  4. Additional information
  5. References

Introduction

Polylysine solutions are typically used to coat glassware and plasticware  for the attachment and growth of cells.

Preparation

  1. Prepare a stock solution by dissolving 100 mg polylysine in 100 ml water (poly-L-lysine or poly-D-lysine can be used; check specific protocol for choice of isomer)and filter sterilize through a 0.22-micron  filter.
  2. Store in 5-ml aliquots at -20°C.

Coating culture dishes, multiwell plates, or chamber slides

  1. When ready to use, dilute 1 part stock solution with 19 parts water to prepare a 50 ?g/ml working solution.
  2. Fill tissue culture dishes, multiwell plates, or slide wells with the working solution and incubate 1 hr in a 37°C incubator, then remove solution by vacuum aspiration and allow surface to dry.
  3. To coat coverslips: Sterilize coverslips by autoclaving or by incubating them in 95% ethanol and drying before coating. Place coverslips in a single layer in a petri dish containing working solution and incubate 1 hr at 37°C. Remove coverslips using sterile forceps and allow surface to dry.
  4. Store coated tissue culture ware up to 3 months at 4°C. Use diluted solutions only once.

Additional information

When coating with poly-l-lysine, the efficiency of coating is relatively low when prepared with distilled water due to the acidic pH of the solution that can range between a pH of 3.3-6.6. One recommendation [1] to improve coating is to use an 8.5  pH borate buffer to dissolve the lysine, with thorough rinsing after coating. In addition to the basic techniques shown here, there are a variety of approaches recommended by vendors as shown below.

VendorRecommendation
MPBio
  1. Dissolve 10mg poly-l-lysine in 1ml water as 1% stock solution.
  2. Dilute stock solution 2 fold in PBS as 1x coating solution.
  3. Add enough coating solution to cover culture surface completely and incubate for 5 minutes at room temperature.
  4. Remove coating solution and rinse 2 times with PBS or serum- free culture medium.
  5. Use immediately or let it dry for future use.
BrainBits
  1. Dissolve poly-d-lysine, (135kd molecular weight), in sterile water to 50ug/ml.
  2. Add 0.15ml/cm² of solution culture surface.
  3. Incubate 1-20 hours.
  4. Rinse 1 time and let dry.
Genlantis
  1. Dissolve 5mg poly-d-lysine, (70-150kd molecular weight), in 100ml sterile water to 50ug/ml.
  2. Add enough poly-d-lysine solution to cover culture surface.
  3. Incubate 1-24 hours.
  4. Remove polylysine solution and rinse the plate thoroughly.
  5. Let the plate dry.
  6. Store coated plates room temperature or 4-8°C
Invitroscience
  1. Dissolve 100mg poly-d-lysine, (70-150kd molecular weight), in 100ml sterile water to 1mg/ml stock solution.
  2. Dilute stock to 0.1mg/ml with sterile water.
  3. Cover culture surface with polylysine coating solution.
  4. Incubate 1 hour at room temperature in hood.
  5. Remove polylysine solution and rinse 3 time with PBS.
Sigma-Aldrich
  1. Add 50ml of sterile water to 5mg of poly lysine.
  2. Coat cell culture surface with 1ml per 25cm² culture surface
  3. Incubate 5 minutes.
  4. Remove poly lysine solution and rinse the plate thoroughly with sterile water and let it dry at least 2 hours.

References

[1] Should I coat coverslips in dd-water? http://www.neuvitro.com/tips-and-calculations-polylysine-coating.htm

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