MAMMALIAN CULTURE MEDIA PREPARATION


Objectives

Prepare the culture media.
Practice sterile technique.
Proliferation Media Formulation: DMEM + 10% FBS + Primocin + 2 ng/ml FGFb. Preparation of solution for the primary cell isolation: DPBS with 10X Anti/Anti

Materials

DPBS with 10X Anti/Anti
Dulbecco's phosphate-buffered saline (DPBS)
100X Antibiotic/Antimyotic
DMEM
Fetal Bovine Serum (FBS)
500X Primocin (antimicrobial)
Fibroblast growth factor beta (FGFb)

Sterile Technique Pointers

Anything that goes into the biohood (including your gloved hands) should be sterilized with 70% ethanol
Don’t block biohood vents with pipette tip boxes, waste boxes, tube racks, etc
Don’t allow liquid to get into the pipette guns/micropipettes
Don’t invert the serological pipette when any liquid is inside, otherwise liquid will flow towards the pipette gun/micropipette and can affect the pipette filter
Later with cells, never spray liquid directly onto the cells (i.e the bottom surface of the flask/well plate). Dispense along the side of well/flask.

Methods

DPBS + Anti/Anti formulation

  1. Take 90 ml of DPBS and add to a 150 ml bottle using a serological pipette
  2. Add 10 ml of 100X Anti/Anti using a serological pipette
    1. Make sure that the anti/anti is homogenous if it was just thawed by pipetting up and down to mix
  3. Store at 4C with your group name + the date + the class

Proliferation Media formulation

  1. Add 17.5 ml of DMEM to a 500 ml bottle of DMEM using a serological pipette.
  2. Add 57.5 ml of FBS to the DMEM bottle using a serological pipette.
  3. Add 1.15 ml of Primocin to the DMEM bottle using a micropipette
  4. Add 11.5 ul of 0.1 mg/ml FGFb to the bottle
  5. Store at 4C with your group name + the date + the class

This protocol was developed for BME 174: cultivated meat laboratory course at Tufts University. The course is administered by Professor David Kaplan's laboratory.








PROTOCOL DEX