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Figure 3. Validation of the autophagosome counting assay. The number of autophagosomes in each image in Figure 2 was determined using a custom-developed image processing sequence for object identification with CellProfiler Software. Rate of autophagosome formation and degradation was successfully monitored with the proposed assay.
) treatment for three hours, followed by removal of the stressors and reestablishment of normoxia in standard growth medium for another 16 hours. An Olympus IX-71 inverted microscope was used for the entire process; all images were taken under the 40x objective.
Figure 5. Images of the LC3-GFP CHO reporter cells taken during each phase of the hypoxia-induced autophagy assay. Cultures were first perfused with standard growth medium under normoxic conditions for 60 minutes, followed by continuous CQ (10 µM, 100 µM, or 1 mM) perfusion in the presence of severe hypoxia (0.2% O2
To simultaneously monitor the two most important organelles involved in autophagy, we further transduced the LC3-GFP reporter CHO cells with a fluorescently tagged, lysosome- specific fusion protein construct, LAMP1-RFP. Transduced cells were incubated under mildly hypoxic conditions (3% O2
) in the presence of CQ at various concentrations for 180
minutes, followed by prolonged 660-minute culture under normoxia in the presence of standard medium.
Figure 4. Assessment of CQ dose response using a dynamic autophagy assay. Three levels of CQ (10 µM, 100 µM, and 1 mM) were perfused through independent culture chambers of the same microfluidic plate at the same time. Time-lapse imaging was performed on three different positions in each of the microchambers. The fluorescence intensity was counted and averaged per frame using CellProfiler Software and normalised to the background to measure flux. Error bars represent standard deviation (S.D.) of the number of counted puncta in approximately 60 cells per time point.
Hypoxia Studies
To further explore the dynamics of stress-induced autophagy, we exploited the CellASIC® ONIX system’s ability to regulate gaseous microenvironments to introduce severely hypoxic conditions within the cell chamber. Prior to analysis, the system’s control of oxygen content was validated. For gas flow rates of 20 mL/min and 3 mL/min, we consistently found that the switch time from normoxic to hypoxic gas environment occurred in less than one hour. For these two gas flow rates, steady-state concentrations were achieved with less than 2% and 10% deviation from the supplied gas, respectively.
In traditional static cultures, achieving equilibrium following defined gas switching is impractical due to incubator size and differences between the measured pericellular oxygen tension (within the flask) and that in the ambient air [8,9]. However, the new platform features a significantly reduced culture vessel size (10,000 cells per chamber) and restricted fluid volume (a few nanolitres), together leading to a faster gas exchange during our hypoxia studies.
Results from initial hypoxia experiments supported this fact; specifically, we found that, compared to the typical hypoxic response of cells cultured in traditional petri dishes [8-12], LC3-GFP CHO reporter cells in the microfluidic perfusion environment were far more sensitive to gas switching, demonstrating autophagosome formation within three hours of hypoxic treatment [10-12]. Following six-hour exposure, a large percentage of cells failed to recover and underwent apoptosis.
Profiling Autophagosome Formation
Based on these preliminary results, we performed dynamic profiling of autophagosome formation in reporter cells in response to CQ (10 µM, 100 µM, or 1 mM) under hypoxia conditions. Similar to results of starvation-induced autophagy, the rate of autophagosome appearance accelerated with respect to increasing CQ dose. As for the recovery phase, cells treated with 100 µM of the CQ responded almost instantaneously, while those treated with the highest dose (1 mM) demonstrated a far more protracted recovery profile (Figure 5 and 6).
Figure 6. Dynamic, quantitative live cell imaging of autophagosome formation with respect to CQ treatment and hypoxia. Three levels of CQ inhibitor (10 µM, 100 µM, and 1 mM) were perfused through independent culture units at the same time. Time-lapse imaging was performed on three different positions in each of the microchambers. The fluorescence intensity was counted and averaged per frame using CellProfiler Software and normalised to the background to measure flux. Error bars represent S.D. of the puncta in around a total of 60 cells per time point.
The data indicate that autophagogome formation started immediately after the switch to hypoxic conditions and lasted for three hours in the cells treated with 1 mM of CQ. In these cultures, lysosome degradation did not occur until almost 11 hours after gas exchange (Figure 7). However, we did not observe any conclusive response in the lysosomal activity during either autophagy or recovery phases except for the observation that lysosomes were instantly condensed under the hypoxic stress.
We speculate that the LAMP1-RFP transduction process (or the LAMP1-RFP construct itself) might be another source of cellular stress, hence affecting overall autophagic activity. We are currently exploring alternative labelling methods for dual-colour assays for hypoxia-induced autophagy.
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