Fast phenotyping of inherited diseases using image cytometry
Characterization for gene therapy at Aarhus University Hospital
To validate the model in patient samples, they use HDFs from patients with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) and the NHDF cell line as a control. Impairment of the VLCAD gene results in failed fatty acid β-oxidation, which causes an increase in the level of reactive oxygen species (ROS) and mitochondrial dysfunction. Patients affected by VLCADD show a range of symptoms, depending on the severity of the disease.
Fernandez’s protocol saves time and requires only small amounts of tissue for each analysis. This approach can be used to develop other cell phenotyping protocols for analyzing customized parameters.
One sample, three parameters analyzed with fast and easy to use assays
The authors use the NucleoCounter® NC-3000™ to study three parameters of HDFs:
1. Cell Number and Viability
The Cell Count and Viability Assay provides information about cell numbers and viability, and is performed in two steps:
- To determine the total cell concentration, cells are lysed, then stained with 4’,6-diamidino-2-phenylindole (DAPI), whereby all nuclei become fluorescent.
- The number of viable cells is determined by adding DAPI directly to untreated cell samples and quantifying them. As DAPI cannot permeate the intact cell membrane, only dead cells that have a compromised membrane stain positive for DAPI.
Viability is calculated by the following formula: Viability% = (Total cell count − dead cell count) / Total cell count × 100.
2. Overall Cellular Redox State
The Cell Vitality Assay provides information about the thiol redox status of cells, and thus the overall cellular redox state, since the processes are linked. This assay makes use of the cell-permeable fluorophore VitaBright-48™ that becomes fluorescent upon reacting with reduced thiol groups on proteins.
3. Mitochondrial Membrane Potential
The Mitochondrial Potential Assay determines the Mitochondrial Membrane Potential (MMP), which is crucial for several aspects of mitochondrial function, and therefore provides information on the overall mitochondrial integrity of cells. The assay uses JC-1, a fluorophore that fluoresces red when it forms aggregates in the mitochondria of healthy cells and fluoresces green when it is dispersed throughout the cytosol in apoptotic cells.
Plug-and-play assays for phenotyping parameters with the NucleoCounter® NC-3000™
The flow chart visualizes the phenotyping protocol in Fernandez’s paper2 . As the initial preparation steps are the same for all three assays, only one sample needs to be prepared.
Approximately 1.6 × 106 HDFs are seeded in a standard T75 cell culture flask. After 24 hours, HDFs are treated with 0, 2 or 4 mmol/l H2O2 for 1, 1.5 and 2 hours, respectively. Next, HDFs are harvested by trypsinization and collected in a 15 ml Falcon sample tube. Four aliquots are taken for cell count, cell viability, thiol redox status and mitochondrial assays, and stained as per the individual assay protocols.
Samples are analyzed using the NC-Slide A2™ with standard protocols for the NucleoCounter® NC-3000™.
To validate the cell phenotyping protocol, HDF tissue samples from VLCADD patients are treated with H2O2 and evaluated.
Oxidative stress is harmful to cells, and H2O2-treatment causes a decrease in both cell numbers and viability (A)2. This correlates with a strong reduction in the mean fluorescence intensity (MFI) of VitaBright-48™, i.e. the amount of reduced thiols (Image B). This is expected, as H2O2 causes oxidation in the intracellular environment. Also, H2O2 treatment induces a marked increase in the number of cells that have lost their mitochondrial membrane potential, measured by the MFI of JC-1 (C), and suggesting a loss of mitochondrial function and initiation of apoptosis in H2O2-treated cells.
Thus, the NC-3000™ assays selected for this cell phenotyping protocol accurately identify cellular and mitochondrial changes in a situation mimicking pathophysiological conditions of elevated ROS levels.
Cellular phenotyping of HDFs from VLCADD patient samples
Next, Fernandez and her team test the response of HDFs from patients suffering either a mild or a severe form of VLCADD. High H2O2 concentration causes a significant decrease in viability in fibroblasts from patients with both forms of the disease compared to the control. This suggests that cells with defective VLCAD are more susceptible to oxidative stress. Interestingly, there is no difference in the intracellular redox state between healthy and sick fibroblasts as evaluated by the Cell Vitality Assay.
Fibroblasts from patients with the mild form of the disease show an increase in the number of cells that lose their MMP, compared to the control. In conclusion, this protocol for cell phenotyping can identify similarities and differences in response to oxidative stress of both healthy and unhealthy HDFs, making it a very efficient method for monitoring mitochondrial function in HDFs of VLCAD patients.
Using image cytometry to develop novel cell phenotyping protocols
The NucleoCounter® NC-3000™ comes with several additional apoptosis assays, a Cell Cycle Assay, and a customizable module called FlexiCyte™, for analysis of up to four different user-defined fluorescent biomarkers. Any of these plug-and-play assays can be incorporated into other cell phenotyping assays as needed. With cell phenotyping protocols using the NC-3000™, only one sample needs to be prepared, which saves time, and less sample volume is required for each phenotyping experiment performed.
References
- P Fernandez-Guerra, M Lund, TJ Corydon et al.: Application of an Image Cytometry Protocol for Cellular and Mitochondrial Phenotyping on Fibroblasts from Patients with Inherited Disorders. JIMD Rep. 2016; 27:17-26.
- Open access to publication and figures through ResearchGate.