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Application of an Image Cytometry Protocol for Cellular and Mitochondrial
Phenotyping on Fibroblasts from Patients with Inherited Disorders
#MMPMID26404456
Fernandez-Guerra P
; Lund M
; Corydon TJ
; Cornelius N
; Gregersen N
; Palmfeldt J
; Bross P
JIMD Rep
2016[]; 27
(?): 17-26
PMID26404456
show ga
Cellular phenotyping of human dermal fibroblasts (HDFs) from patients with
inherited diseases provides invaluable information for diagnosis, disease
aetiology, prognosis and assessing of treatment options. Here we present a cell
phenotyping protocol using image cytometry that combines measurements of crucial
cellular and mitochondrial parameters: (1) cell number and viability, (2) thiol
redox status (TRS), (3) mitochondrial membrane potential (MMP) and (4)
mitochondrial superoxide levels (MSLs). With our protocol, cell viability, TRS
and MMP can be measured in one small cell sample and MSL on a parallel one. We
analysed HDFs from healthy individuals after treatment with various
concentrations of hydrogen peroxide (H2O2) for different intervals, to mimic the
physiological effects of oxidative stress. Our results show that cell number,
viability, TRS and MMP decreased, while MSL increased both in a time- and
concentration-dependent manner. To assess the use of our protocol for analysis of
HDFs from patients with inherited diseases, we analysed HDFs from two patients
with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD), one with
a severe clinical phenotype and one with a mild one. HDFs from both patients
displayed increased MSL without H2O2 treatment. Treatment with H2O2 revealed
significant differences in MMP and MSL between HDFs from the mild and the severe
patient. Our results establish the capacity of our protocol for fast analysis of
cellular and mitochondrial parameters by image cytometry in HDFs from patients
with inherited metabolic diseases.