Optimization of Fluorescence Assay of Cellular Manganese Status for High Throughput Screening

Abstract

The advent of high throughput screening (HTS) technology permits identification of compounds that influence various cellular phenotypes. However, screening for small molecule chemical modifiers of neurotoxicants has been limited by the scalability of existing phenotyping assays. Furthermore, the adaptation of existing cellular assays to HTS format requires substantial modification of experimental parameters and analysis methodology to meet the necessary statistical requirements. Here we describe the successful optimization of the Cellular Fura-2 Manganese Extraction Assay (CFMEA) for HTS. By optimizing cellular density, manganese (Mn) exposure conditions, and extraction parameters, the sensitivity and dynamic range of the fura-2 Mn response was enhanced to permit detection of positive and negative modulators of cellular manganese status. Finally, we quantify and report strategies to control sources of intra-and inter-plate variability by batch level and plate-geometric level analysis. Our goal is to enable HTS with the CFMEA to identify novel modulators of Mn transport.

Cellular manganese content is developmentally regulated in human dopaminergic neurons

“…Our laboratory had previously developed the Cellular Fura-2 Mn Extraction Assay (CFMEA) to enable rapid fluorescent-based measurements of intracellular Mn content 26 . Here, we utilized CFMEA for high throughput screening (HTS).…”

“…The overall HTS approach consisted of pretreatment with the test small molecule, followed by co-exposure to 125 µM MnCl 2 , representative of in vivo brain Mn levels at which aberrant function would be observed 23 . An immortalized murine striatal neuron lineage (STHdh Q7/Q7 ) was used in the HTS, as the striatum, like other structures of the basal ganglia, has a relatively high basal Mn level and an especially high capacity for Mn accumulation 26 27 . After the Mn and small molecule incubation period, extracellular Mn was washed away and CFMEA performed ( Fig.…”

Untargeted metabolic profiling identifies interactions between Huntington's disease and neuronal manganese status

“…The clonal striatal cell lines—both mutant STHdh[Q111/Q111] and wild-type STHdh[Q7/Q7] were grown at 33°C 31 . Culture and exposures were performed as previously described 21, 32 . Subsequently, STHdh[Q111/Q111] and wild-type STHdh[Q7/Q7] cells were plated at equal density 16 hours before treatment.…”

Manganese-Induced Parkinsonism and Parkinson’s Disease: Shared and Distinguishable Features

“…The paramagnetic nature of Mn allows for detection and examination of Mn trafficking dynamics and pharmacokinetics using sensitive and non-invasive methods including positron emission tomography (PET), single-photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI) [ 61 , 62 , 63 , 64 ]. Other analytical methods including atomic absorption spectroscopy (AAS), atomic emission spectroscopy (AES), inductively coupled plasma-atomic emission spectrometry (ICP-AES), mass spectrometry (ICP-MS), neutron activation analysis, X-ray fluorometry, spectrophotometry, and radioactive trace assay and cellular fura-2 manganese extraction assay (CFMEA) are used to measure Mn levels in biological specimens [ 65 , 66 , 67 ]. Chronic high- (>1 mg/m 3 ) and low-levels (0.5–1.0 mg/m 3 ) of Mn inhalation exposures in the workplace have been reported to result in Mn accumulation in the brain and cause Mn-induced parkinsonism and subtle subclinical changes in the general population respectively [ 68 , 69 , 70 , 71 , 72 ].…”