Cell phenotyping protocol for characterizing inherited diseases using image cytometry
-Easy and quick characterization of cellular and mitochondrial parameters in your cells
Cellular phenotyping of human dermal fibroblasts derived from patients with inherited diseases can be a very strong tool to understand various aspects of a disease, including aetiology. In a recent study, Fernandez et al have developed a novel cell phenotyping protocol using the NC-3000™ image cytometer to characterize cellular and mitochondrial parameters in human dermal fibroblasts (HDFs). Their protocol is time saving and requires small amounts of sample for each analysis. A similar approach can be used to develop other cell phenotyping protocols, where the cellular parameters measured is completely customizable. Below we will go through which cellular parameters were measured in this protocol, and how it performed under conditions mimicking a pathophysiological situation.
Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) as a model system to develop a novel cell phenotyping protocol
As a model to develop the new cell phenotyping protocol, the authors used human dermal fibroblasts (HDFs). As a control, the NHDF cell line was used and for disease study, HDFs from patients with very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) were analysed. Impairment of the VLCAD gene results in failed fatty acid β-oxidation, which causes an increase in reactive oxygen species (ROS) levels and mitochondrial dysfunction. Patients affected by VLCADD show a range of symptoms, depending on the severity of the disease.
One sample – three parameters analyzed with straight forward plug and play assays
The authors decided to use the NC-3000™ to study human dermal fibroblasts with three different parameters:
1) Cell concentration and viability – Provides information on cell proliferation and death.
The assay was performed in two steps. First, to determine the total cell concentration, cells were lysed and intact nuclei were incubated with DAPI, a non-permeable fluorophore that becomes fluorescent once bound to DNA. Second, the number of dead cells was determined by adding DAPI directly to cell samples without them being lysed. As DAPI is not permeable to the cell membrane, only dead cells stain positive for DAPI. Viability is calculated by the following formula:
Viability% = ((Total Cell Count – Dead Cell count) / Total Cell Count) * 100
2) Thiol redox status (TRS) – The thiol redox status gives information of the overall cellular redox state, as both processes are linked.
Determined using VitaBright-48TM, a cell-permeable fluorophore that becomes fluorescent upon reacting with thiol groups on proteins.
3) Mitochondrial membrane potential (MMP).
MMP is crucial for several aspects of mitochondrial function, thus providing information on the overall mitochondrial integrity. Determined using JC-1, a fluorophore that fluoresce red when it forms aggregates in the mitochondria of healthy cells, and fluoresce green when it is dispersed throughout the cytosol in apoptotic cells.
Plug and play assays to evaluate all these markers comes with the NC-3000™ system. As the initial preparation steps are the same for all three assays, only one sample needs to be prepared.
Validating the Cell Phenotyping Protocol
To validate the cell phenotyping protocol, human dermal fibroblasts were treated with H2O2 and evaluated for the above three parameters. In agreement with the notion that oxidative stress is harmful to cells, H2O2 treatment caused a decrease in both viability and cell concentration (fig 2A). This correlated with a strong reduction in the number of reduced thiols as measured by VitaBright-48TM, which is expected as H2O2 causes an oxidation of the intracellular environment (Fig 2B). Also, H2O2 treatment induced a marked increase in the number of cells that had lost their mitochondrial membrane potential, suggesting a loss of mitochondrial function and initiation of apoptosis in treated cells (Fig 2C). Thus the NC-3000™ assays selected for this cell phenotyping protocol could successfully identify cellular and mitochondrial changes in a situation mimicking pathophysiological conditions with elevated ROS levels.
Fig. 2 Effect of exogenous H 2 O 2 treatment for different time periods in two different NHDF cell lines. NHDFs were treated with 0, 2 or 4 mmol/L H 2 O 2 for 1, 1½ and 2 h. (a) Viability quantification. Cell viability was quantified by counting DAPI-positive cells in the total cell population. (b) Thiol redox status (TRS). HDFs were incubated with VB-48, AO and PI. VB-48 was used to determine the levels of free thiols, AO as a nuclear staining and PI to label dead cells (removed from the analysis). The values shown correspond to the mean fluorescence intensity (MFI). (c) Mitochondrial membrane potential (MMP). HDFs were incubated with JC-1 to establish the percentage of cells with a depolarized MMP, and dead cells were excluded by DAPI staining.
Cellular phenotyping of human dermal fibroblasts with patients with VLCADD
Next the authors tested the response of human dermal fibroblasts derived from patients suffering either a mild or severe form of VLCADD. High H2O2 concentration caused a significant decrease in viability in fibroblasts from patients with both the mild and the severe form of the disease compared to the control. This suggests that cells with defective VLCAD are more susceptible to oxidative stress. Interestingly, there was no difference in the intracellular redox state between healthy and sick fibroblasts as evaluated by the VitaBright-48TM assay. Interestingly, fibroblasts from the patient with the mild form of the disease, showed an increase in the number of cells that lost their MMP compared to the control. Overall, the cell phenotyping protocol was able to identify both similar and different responses to oxidative stress of both healthy and sick human dermal fibroblasts.
Using image cytometry to develop novel cell phenotyping protocols
Here Fernandez et al has shown that image cytometry can be used to develop novel cell phenotyping protocols to study the molecular pathological mechanism of human dermal fibroblasts derived from patients with inherited diseases. Using three plug and play assays from the NC-3000™ system, they could successfully characterize phenotypic changes in fibroblast grown under conditions mimicking pathophysiological conditions. Also they identified differences between fibroblasts derived from healthy individuals and patients with VLCADD.
The NC-3000™ also comes with several additional apoptosis assays, cell cycle assays, and a customizable module called FlexiCyte, that allows for the analysis of up to 4 different fluorescent biomarkers. Any of these plug and play assays could be incorporated into other cell phenotyping assays as needed. With cell phenotyping protocols on the NC-3000™, only one sample needs be prepared, thus saving time and requiring less sample for each cellular phenotyping experiment performed.