The effect of foetal bovine serum supplementation upon the lactate dehydrogenase cytotoxicity assay: Important considerations for in vitro toxicity analysis.
The lactate dehydrogenase (LDH) assay is a commonly-used tool for assessing toxicity in vitro. However, anecdotal reports suggest that foetal bovine serum (FBS) may contain LDH at concentrations significant enough to interfere with the assay and thus reduce its sensitivity. A series of experiments were performed to determine whether addition of FBS to culture medium significantly elevated culture media LDH content, and whether replacement of FBS with heat inactivated foetal bovine serum (HI-FBS) reduced LDH content and interfered with cell response to cytotoxic challenge. The addition of FBS at 5, 10 and 15% final concentrations increased culture medium LDH content in a dose-dependent manner. The substitution of HI-FBS for FBS reduced culture medium LDH content and increased the dynamic range of the assay. Cell viability of the SH-SY5Y human neuroblastoma and N27 rat mesencephalic neurone cell lines were significantly reduced as measured using the MTT reduction assay, whilst HI-FBS only affected toxicity response in a cell- and toxin-specific manner, although these effects were small. Hence, for cell lines with a high FBS requirement, the use of HI-FBS or alternative toxicity assays can be considered, or the use of alternative formulations, such as chemically-defined serum-free media, be adopted.
King's College London, Institute of Pharmaceutical Science, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom.
Martin G
MG
Thomas
King's College London, Institute of Pharmaceutical Science, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom.
Roxanne M
RM
Marwood
King's College London, Institute of Pharmaceutical Science, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom.
Anna E
AE
Parsons
King's College London, Institute of Pharmaceutical Science, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom. Electronic address: richard.parsons@kcl.ac.uk.
Richard B
RB
Parsons
D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D002243:Carbolines / Q000633:toxicity; D002417:Cattle; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000032:analysis*; D009536:Niacinamide / Q000031:analogs & derivatives / Q000633:toxicity; D051381:Rats; D018675:Toxicity Tests / Q000379:methods*
D002243:Carbolines; D003470:Culture Media; D009536:Niacinamide; D000255:Adenosine Triphosphate; C010262:norharman; D007770:L-Lactate Dehydrogenase; C024058:N(1)-methylnicotinamide
Assay interference; Cell viability; Foetal bovine serum; Heat inactivated; Lactate dehydrogenase; MTT assay