In this study, our initial bulk-cell analysis results also showed a slightly higher but statistically not significant average mtDNA copy quantity in CP-C cells (1,530 per cell) as compared to CP-A (1,392 per cell) (Fig. led to the postulate that mitochondria, the primary target for oxidative damage, can function as an endogenous oxygen sensor. Probably one of the most important factors determining drug response and aggressiveness of tumors is the large intratumoral heterogeneity. PPP1R53 Recent studies have shown that actually cells inside a clonal human population or seemingly homogenous tissue show considerable variability of different characteristics ranging from gene manifestation levels to phenotypic features [20]C[22]. It is right now broadly approved that mitochondrial heterogeneity, including variations in mtDNA copy quantity, DNA mutation/depletion, manifestation and rules of genes encoded by mtDNA, and activity levels, is an important contributor to mitochondrial difficulty and contributes to the overall cell-cell heterogeneity [23]C[25]. Most current bioanalytical techniques collect data using thousands to millions of cells, inherently providing results averaged over a large cell human population. Such bulk-cell methods could potentially miss important and valuable info when dealing with highly heterogeneous systems [26] such as AM 114 cancer [27]. Consequently, the development and software of techniques capable of carrying out analyses in the single-cell level are essential, not only for a better understanding of core cellular processes, but also for new, more effective strategies for disease prevention, management, and treatment [28]C[31]. With this study we use two immortalized human being Barretts esophageal epithelial cell lines CP-A and CP-C that were originally derived from individuals with Barretts esophagus (Become) without dysplasia and with dysplasia, respectively [32]. Although both are nonmalignant epithelial cells, it was found that CP-C cells were more resistant to oxidative stress induced by bile acid (chenodeoxycholic acid (CDCA)) than CP-A, suggesting that, at least with regard to acid response, CP-C cells behave more like esophageal malignancy cell lines as compared to CP-A cells [2]. In this study, we aim to elucidate potential mechanisms leading to malignant transformation in Become by quantifying variations in the way cells respond to the oxidative stress caused by hypoxia. We have applied a qPCR-based technique developed in our lab to determine the mtDNA copy number and the manifestation levels of mitochondrial and nuclear genes in individual cells. Utilizing single-cell analysis we distinguished variations in mtDNA copy quantity, mitochondrial membrane potential, and hypoxia response gene manifestation levels between CP-A and CP-C cells which cannot be expected by bulk cell analysis. The application of these fresh methods, along with single-cell O2 usage measurements [33]C[35], allowed the characterization of delicate hypoxia response variations between CP-A and CP-C cells. A better understanding of the molecular basis of EAC initiation and development will facilitate attempts to define potential restorative targets. Materials and Methods Cell Tradition and Hypoxia Treatment The Barretts esophageal epithelial cell lines CP-A and CP-C were from ATCC and cultivated in AM 114 Gibco? Keratinocyte Serum-Free Medium (SFM) cell growth medium (Invitrogen, Carlsbad, CA), supplemented with hEGF (Peprotech, Rocky Hill, NJ) at 5.0 g/L, BPE (bovine pituitary extract) at 50 mg/L and penicillin/streptomycin solution (Invitrogen, Carlsbad, CA) at 100/100 g/mL inside a tissue-culture incubator at 37C in humidified air with 5% CO2. Prior to experiments, cells were cultured inside a 75 cm2 AM 114 flask to approximately 80% confluence. Cells in G1 phase sorted AM 114 with FACSAria (BD Biosciences, San Jose, CA) were used in qPCR experiments in this study. For hypoxia, CP-A and CP-C cells at 80% confluence were incubated in the keratinocyte SFM medium comprising 2% (v/v) Oxyrase (Oxyrase, Inc., Mansfield, OH) at 37C for 30 minutes, which is the ideal Oxyrase treatment time mainly because identified previously [31]. The cells were consequently trypsinized in 0.05% (v/v) trypsin solution containing 2% (v/v) Oxyrase at 37C for 9 min. The trypsinization was clogged by adding Dulbeccos Modified Eagle Medium (DMEM) (Invitrogen) supplmented with 5% fetal bovine serum (FBS) (Invitrogen) comprising 2% (v/v) Oxyrase. Single-cell Harvesting Single-cell harvesting (aspiration and dispensing) was performed using a micromanipulator developed by our group [36], [37] (Methods S1). Primer Design and Selection of Gene Target Fragments within the hypervariable region I (HVI) in mtDNA were chosen for copy number analysis [38], [39]. Total DNA isolated from bulk samples (1104 cells) was used as template for mtDNA copy number measurement, and quantified using a Real-Time qPCR System (StepOne, Applied Biosystems, Foster City, CA) using optimized primers (Methods S1). For RT-qPCR manifestation level analysis, four mitochondrially encoded genes (16s rRNA, and four nuclear genes (28s rRNA, VEGF, MT3, and PTGES)(primers sequences as [31]) were chosen (Methods S1). Single-cell mtDNA Copy Number Dedication After harvesting, tubes each comprising one cell suspended in.