REQUIRED LABORATORY EQUIPMENT

 

  • Sterile laminar flow cell culture cabinet for cell culture manipulation and set up.
  • Cell culture incubator kept at 37°C, in a highly humidified atmosphere of 95% air and 5% CO2.
  • CO2 gas tank.
  • Water bath, 37°C.
  • Aspiration System.
  • TEER measurement equipment: Millicell©, ref.ERS2 or WPI, ref.EVOM2.
  • Fluorimeter and associated plates to measure (485nm excitation, 527nm emission).
  • Multichannel micropipettes (volume range: 50-1200 µL).
  • Automatic Pipettes (volume range: 20-1000 µL).
  • Sterile culture medium containers (for example: COSTAR 50 ml #4870).
  • Disposable Pippette tips.
  • Bioanalytical capacity (Mass Spectrometry).

 

Pipetas

 

 

REAGENTS AND SOLUTIONS

 

Standard Caco-2 cell culture medium: EMEM (EBSS) + 2mM Glutamine + 1% Non-Essential Amino Acids (NEAA) + 10% to 20%  Foetal Bovine Serum FBS / FCS.

HBSS (10X) buffer: Adjust to pH ~ 7.4, autoclave and store at 4°C until use.

CaCl2 2H2O (100 mM), autoclave and store at 4°C until use.

MgCl2 2H2O (50 mM), autoclave and store at 4°C until use.

Ethanol (70%).

Lucifer Yellow Stock 1mg/mL (SIGMA #L0259/#62642).

 

 
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ready-to-use-technology

 

 

The final HBSS (1X)+Ca2+/Mg2+ buffer should be prepared from stock solutions the same day of the experiments.

Alternatively, commercial HBSS transport buffer can be purchased from SIGMA (REF: H8264).