(Fig. 1A) Cells and neutralized collagen were mixed and pipetted into one well. After 15 min incubation at 37°C a hydrogel is formed. Medium was absorbed from the hydrogel (Fig. 1B) to increase concentration of collagen and cells to in vivo conditions. The process is completed (Fig. 1C) in less than 1 hour and results in a structure that is about 120 μm thick.
Oxygen Consumption Measurements
Culture medium was removed after a desired culture period and 100 μl of MitoXpress Xtra stock solution prepared in prewarmed DMEM was added. 1 μl of compound (100x) was added to appropriate wells. All wells were then sealed by adding 100 μl prewarmed HS mineral oil to prevent the back diffusion of ambient oxygen. The plate was then measured kinetically on a FLUOstar Omega for 90-120 minutes at 37°C.
Extracellular Acidiﬁcation Measurements
Two hours prior to measurement the RAFT cell culture plate was placed in a CO2 free incubator at 37°C, 95% humidity, in order to remove CO2 from the plate material. Media was removed and 2 wash steps were performed using the Respiration Buffer (0.5 mM KH2PO4, 0.5 mM K2HPO4, 20 mM Glucose, 4.5 g/L NaCl, 4.0 g/L KCl, 0.097 g/L MgSO4, 0.265 g/L CaCl2). Finally 150 μl of Respiration Buffer containing pH-XtraTM probe at the recommended concentration was added to each well. The plate was then measured kinetically.
FLUOstar Omega/CLARIOstar® instrument settings
|Measurement method:||Time-resolved fluorescence (Omega: TR-F Optical attachment installed)|
|Measurement mode:||Plate Mode Kinetic|
|Measurement time:||120 min (data points every 2 min)|
Dual chromatic using the following windows:
|Excitation/ Emission filters||Integration start/time (µs)|
1 TREx L + 655-50 (Omega) + 645-20 (CLARIOstar)
2 TREx L + 655-50 (Omega) + 645-20 (CLARIOstar)
|pH-XtraTM Glycolysis Assay|| |
1 TREx L and 615-BP10 100/40
2 TREx L and 615-BP10
Results & Discussion
Sample oxygen consumption profi les are presented in Figure 2.
Metabolic processes like glycolysis and respiration are essential functions that shed light on cellular health and should be monitored in response to potential drugs. The MitoXpress® Xxtra HS assay from Agilent Technologies uses an O2 sensitive fluorophore for measuring oxygen consumption which correlates with mitochondrial function. The pH-Xtra™ assay uses a pH-sensitive fluorophore which detects acidification due to glycolysis-related release of lactate. Recently, 3D cell cultures aim at bridging between conventional cell culture at in vivo studies.
Increases in oxygen consumption were determined for increasing cell densities as well as upon addition of the mitochondrial uncoupler FCCP. Furthermore, modulation of lactate production by addition of oxamate or antimycin led to lower or higher acidification, respectively, as detected with pH-Xtra on FLUOstar® Omega.