Assessment of Extracellular Acidification using a Fluorescence-Based Assay

James Hynes¹, Conn Carey¹, Sinead Kriwan¹, Catherine Wark²,
¹ Luxcel Biosciences Ltd., Cork, Ireland, ² BMG LABTECH Aylesbury, U.K.

Time-Resolved Fluorescence assay to measure Extracellular Acidification
Measures the conversion of Pyruvate to Lactic Acid
Convenient measure of glycolytic flux

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Introduction

Metabolic perturbations play a critical role in a variety of disease states and toxicities. Knowledge of the interplay between the two main cellular ATP generating pathways; glycolysis and oxidative phosphorylation, is therefore particularly informative when examining such perturbations. Here we describe a time resolved fluorescence-based assay for the assessment of Extracellular Acidification which provides data on the rate of conversion of pyruvate to lactic acid and is therefore a convenient measure of glycolytic activity. Such assays are particularly informative when assessing alterations in glucose metabolism, detecting glycolytic inhibition and as a confirmatory analysis in the identification of mitochondrial dysfunction. The following protocol outlines how such measurements can be carried out on standard microtitre plates using the fluorescent pH-sensitive probe, pH-Xtra and ratiometric time resolved fluorescence detection. This approach overcomes the calibration and biocompatibility issues associated with some existing probes thereby allowing conventional cell culturing and assay procedures whilst also facilitating accurate quantitative analysis. In addition, spectral compatibility with MitoXpress®, the Luxcel oxygen sensitive probe, facilitates a multiplexed measurement approach providing a comprehensive metabolic assessment of test cells.

Materials and Methods

Clear 96 well plates from Costar
pH-Xtra probe from Luxcel Biosciences, Ireland
FLUOstar Omega microplate reader from BMG LABTECH

Plate Preparation and Reading
Warm instrument to measurement temperature (typically 30°C). Prepare kinetic measurement protocol to read the plate at 2-4 min intervals over 1-2 h period using the recommended ratiometric measurement parameters (more detail in the "Measurement" section). Adherent cells were either (A) plated at the indicated concentration in L15 medium and cultured in CO₂-free conditions, 95% humidity at 37°C overnight or (B) cultured overnight in a standard CO₂ incubator and then maintained in CO₂-free conditions for 2.5 h prior to the measurement.(1)
Reconstitute pH-Xtra probe in 1 mL of Millipore water. Warm to measurement temperature. Wash cells with measurement buffer(2) being careful not to dislodge cells from the base of the wells. Add 150 µL of the pre-warmed measurement buffer to each well and place the plate on a plate heater equilibrated to 30°C.
Using a repeater pipette, add 10 µL pH-Xtra probe to each well. Follow with drug addition if applicable. Oil can be added if bulk acidification data is required.(3)

Measurement and Data Analysis
Insert the microplate into the FLUOstar Omega. Glycolytic activity is measured using the BMG LABTECH scripting function. pH-Xtra probe is measured using dual delay, time-resolved measurements. Optimal filter wavelengths are 340 TR L for excitation and TR 615 for emission. Delay times of 100 and 300 µs are used, both with a measurement window of 30 µs respectively. Plate preparation time should be kept to a minimum. When the measurement cycle is completed, remove the plate from the instrument and save the measured data to a file. These dual intensity measurements are used to calculate emission lifetime using the following function, τ=t2-t1/ Ln (D2/D1) [t=delay time, D=measured intensity value]. Scripts and protocols can be obtained through your local BMG LABTECH representative and MARS templates are available to facilitate data analysis.

Fig. 1: Summary of pH-Xtra Assay showing a general schematic of assay preparation (A). Cellular metabolic processes (B) cause an acidification of the media resulting in an increase in probe signal (C). If necessary, this signal can be conveniently converted into a H⁺ scale using a MARS template (D).

Results and Discussion

Monitoring Cellular Respiration
The ability of the pH–Xtra assay to assess cell respiration is illustrated in Fig. 2. A typical data read out is presented in Fig 2A showing the parallel analysis of 96 individual samples. A serial dilution of HepG2 is presented in Fig 2B with increasing cell numbers causing an increased rate of acidification. This is seen as in increased rate of signal change. Profiles are highly reproducible showing %CV values of the order of <5%.

Fig. 2: A) 96 well plate based analysis of extracellular acidification measured on a FLUOstar Omega microplate reader, cell profiles on left, control profiles on right). B) Acidification profiles for HepG2 cultured in L15 medium at the indicated seeding concentration (cells/well).

Monitoring Perturbed Metabolism
Treatment with 2DG results in reduced glucose uptake due to competitive inhibition of glucose transport. Treated cells show dramatic, immediate and dose dependant decreases in rates of extracellular acidification (Fig 3A).
Mitochondrial toxins such as electron transport chain inhibitors prevent or restrict aerobic ATP generation. In many cell systems this leads to an increase in glycolytic flux to supply cellular ATP. This increased acidification can therefore be used as a confirmatory parameter when investigating drug-induced mito-chondrial dysfunction. These rates are typically compared to untreated cells to determine the direction and magnitude of the effect observed. Such response can be seen in Fig 3B where treatment with Sodium Oxamate and 2DG cause the expected decreases in extracellular acidification while treatment with the Electron Transport Chain inhibitor Antimycin causes a significant increase in acidification in an attempt to maintain cellular ATP supply. This assay can be multiplexed with the Luxcel MitoXpress® probe to assess both Extracellular Acidification and Oxygen Consumption on the same plate.

Fig. 3: A) Effect of increasing concentrations of 2DG on measured HepG2 acidification. Data are mean ±SD, n=4. B) Acidification profiles of HepG2 cells illustrating the effect of compound treatment

Conclusion

Analysis of cellular glycolytic flux is possible on standard 96 well plates by measuring rates of extracellular acidification.
The ratiometric measurement approach facilitated by the pHXtra probe and FLUOstar Omega provides a robust assay readout.
Such measurements allow convenient cellular metabolic characterisations.
When assessed alongside an analysis of oxygen consumption (see application note 207) a detailed analysis of mitochondrial function is possible
Data analysis is greatly simplified using MARS data analysis templates.

Fig. 4: BMG LABTECH's FLUOstar Omega multidetection microplate reader

Supplemental Information

When culturing cells in a standard 5% CO₂ environment the polystyrene body of a standard microtitre plate absorbs CO₂. If a plate is moved immediately from 5% CO₂ to the measurement chamber this CO₂ will diffuse into the sample causing significant acidification. To circumvent this, cells may be cultured in CO₂-free conditions in specially formulated media such as L-15, or cultured in standard media and moved to a CO₂-free environment 2h prior to measurement.
Measurement Buffer 1: 1mM K-phosphate, 20mM Glucose, 0.07M NaCl and 0.05M KCl, 0.8mM, MgSO4, 2.4mM CaCl2, pH 7.4. Measurement Buffer DMEM Base, 1.85g/l NaCl, 10ml 100x GlutaMax, 10mL 100mM Sodium Pyruvate, 15 mg Phenol Red, 25mM glucose.
Acidification in unsealed samples is due almost entirely to lactate conversion while, in sealed samples there is a strong contribution from carbonic acid due to trapped CO₂ reflecting Krebs cycle activity.