Making Complete Culture Media

Youssef Farhat, Written 2/20/12, Last updated 5/6/14

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Introduction

This protocol is for making complete media suitable for culturing tenocytes (tendon cells).  It contains MEM α, 20% FBS, 1% Pen-Strep, and a small amount of 2-Mercaptoethanol.  Basal media (MEM α) provides the basic nutrients that cells need, such as amino acids, ions, and a pH buffer.  FBS contains proteins, lipids, and growth factors that support cell growth.  Pen-Strep acts as an antibiotic, preventing bacterial contamination.  2-mercaptoethanol serves as an antioxidant, protecting cells and proteins from oxidative harm.

Materials

  • MEM a (Gibco, #12561)
  • FBS (Sigma-Aldrich, #F6178)
  • Pen-Strep (Gibco, #15140)
  • 2-Mercaptoethanol (Sigma-Aldrich, #M3148)
  • Sterile-filter
  • Sterile bottle or container
  • Pipettor and 10 µL micropipettor
  • Serological pipettes and 10 µL pipette tips
  • 70% EtOH
  • 1.5 mL Eppendorf tube

Procedure

1)     After completely thawing the FBS, heat it for 30 min at 56°C to inactivate complement (unwanted blood clotting) proteins.

2)     Check the hood to be sure that the blower has been on for at least 15 minutes.

3)     Wear gloves and a lab coat while working inside of the hood.

4)     Place the following inside of the hood, spray with 70% EtOH and wipe them with paper towels or a Kimwipe:

  • MEM α, FBS, Pen-Strep, 2-Mercaptoethanol
  • Sterile-filter, sterile bottle or container
  • Pipettor and 10 µL micropipettor
  • 10 µL pipette tips
  • 70% EtOH
  • 1.5 mL Eppendorf tube

5)     Spray all materials and the entire hood surface with 70% Ethanol and wipe dry.

6)     Using sterile technique, attach the sterile filter to the bottle.  Then, attach the vacuum to the filter nozzle and turn the vacuum on.

7)     Combine the following (in order) through the vacuum filter:

  • 10 mL of Pen-Strep
  • 200 mL of FBS
  • 790 mL of Media (MEM α)

8)     Remove the sterile filter from the bottle.

9)     Add 6.5 µL of 2-Mercaptoethanol to the media and promptly pop the tip off into the 1.5 mL Eppendorf tube and close the lid to avoid stinking up the lab.

10)  Label your container with the following information:

  • Name
  • Date
  • Tenocyte Media
  • MEM a + 10% FBS + 1% PS + 6.5 µL/L of 2-ME

 

Citing this Protocol

Farhat, Y. “Making Complete Culture Media.” Protocol Place. March 2012. Accessed on mm/dd/yyyy. <http://protocol-place.com/>

 

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