Plate reader-based assays for measuring cell viability, neuroprotection and calcium in primary neuronal cultures

Abstract

Drug discovery and development efforts critically rely on cell-based assays for high-throughput screening. These assay systems mostly utilize immortalized cell lines, such as human embryonic kidney cells, and can provide information on cytotoxicity and cell viability, permeability and uptake of compounds as well as receptor pharmacology. While this approach has proven extremely useful for single-target pharmacology, there is an urgent need for neuropharmacological studies to screen novel drug candidates in a cellular environment resembles neurons in vivo more closely, in order to gain insight into the involvement of multiple signaling pathways. Primary cultured neuronal cells, such as cortical neurons, have long been used for basic research and low-throughput screening and assay development, and may thus be suitable candidates for the development of neuropharmacological high-throughput screening approaches. We here developed and optimized protocols for the use of primary cortical neuronal cells in high-throughput assays for neuropharmacology and neuroprotection, including calcium mobilization, cytotoxicity and viability as well as ion channel pharmacology. Our data show low inter-experimental variability and similar reproducibility as conventional cell line assays. We conclude that primary neuronal cultures provide a viable alternative to cell lines in high-throughput assay systems by providing a cellular environment more closely resembling physiological conditions in the central nervous system.

An optimized lactate dehydrogenase release assay for screening of drug candidates in neuroscience

“…In order to quantify the level of oxidative stress we used the fluorescent ROS indicator 6-carboxy-2′, 7′ dichlorodihydrofluorescein diacetate (DCFDA) as described by us previously (Burroughs et al, 2012). Briefly, cells were loaded with 10 μM DCFDA in complete media for 30 min.…”

“…The MTT assay was performed essentially as described by us previously for HT-22 cells and primary cortical neuron culture (Burroughs et al, 2012; Kaja et al, 2011). Briefly, media was aspirated from the cells and replaced with 100 μl of 1.2 mM MTT in HBSS with calcium and magnesium (Lonza, Walkersville, MD) supplemented with 10 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) at pH 7.3.…”

Plate reader-based cell viability assays for glioprotection using primary rat optic nerve head astrocytes

“…ROS levels due to t BHP treatment were quantified using the membrane-permeable, fluorescent ROS indicator DCFDA essentially as previously described by us for neurons (Burroughs et al, 2012). ONHAs were seeded in 96-well plates were loaded with 10 μM DCFDA in complete media for 45 min and oxidative stress was chemically induced as described above.…”

“…The calcein-AM uptake assay was performed essentially as described previously for primary culture of cortical neurons (Burroughs et al, 2012). Briefly, cells in 96-well plates were loaded with 5 μM calcein-AM (Life Technologies, Carlsbad, CA) in growth media from a 5 mM stock dissolved in DMSO for 1 hour at 37 °C/5% CO 2 /95% humidity.…”

“…The MTT assay was performed essentially as described by us previously for C6 astroglia cells, HT-22 cells, and primary cortical neuron culture (Burroughs et al, 2012; Kaja et al, 2011; Kaja et al, 2015b). Briefly, the medium was aspirated from the cells and replaced with 100 μl of 1.2 mM MTT in HBSS with calcium and magnesium (Lonza, Walkersville, MD) supplemented with 10 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) at pH 7.3.…”