Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration.
We describe a simple protocol for determining the oxygen consumption of cells in static culture. The protocol is based on a noninvasive oxygen-sensing microplate and a simple mathematical model derived from Fick's Law. The applicability of the model is confirmed by showing the correlation of computed oxygen consumption rate (OCR) values to actual cell densities ascertained by direct cell counting and/or MTT for HL60 and U937 cells cultured in suspension. Correlation between computed OCR and these other indications of cell number was quite good, as long as the cultures were not diffusion-limited for oxygen. The impact of the geometric factors of media depth and well size were confirmed to be consistent with the model. Based on this demonstrated correlation, we also developed a simple, completely noninvasive algorithm for ascertaining the per-cell oxygen utilization rate (OUR), which is the ratio of OCR to cell number, and a fundamental cell characteristic. This is accomplished by correlating the known seed densities to extrapolated determinations of OCR at time zero. Such determinations were performed for numerous cell types, in varying well sizes. Resulting OUR values are consistent with literature values acquired by far more painstaking methods, and ranged from <0.01 fmol.min(-1).cell(-1) for bacteria to 0.1-10 fmol.min(-1).cell(-1) for immortalized mammalian and insect cell lines to >10 fmol.min(-1).cell(-1) for primary hepatocytes. This protocol for determining OCR and OUR is extremely simple and broadly applicable and can afford rapid, informative, and noninvasive insight into the state of the culture.
Guarino
Richard D
RD
BD Technologies, Research Triangle Park, North Carolina.
Dike
Laura E
LE
Haq
Tariq A
TA
Rowley
Jon A
JA
Pitner
J Bruce
JB
Timmins
Mark R
MR
D000818:Animals; D001681:Biological Assay; D015374:Biosensing Techniques / Q000379:methods*; D002452:Cell Count; D018929:Cell Culture Techniques; D002460:Cell Line; D002470:Cell Survival; D006801:Humans; D008832:Microchemistry / Q000379:methods*; D010100:Oxygen / Q000032:analysis; D010101:Oxygen Consumption; D013778:Tetrazolium Salts / Q000032:analysis; D013844:Thiazoles / Q000032:analysis
D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue; D010100:Oxygen