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.

Figure 1. Cell viability and neuroprotection assays using primary cultured cortical neurons in 96 well-plate assays

(A) Representative images of primary cortical culture at 5 days in vitro. Cells differentiation was obvious by significant connections and dendrite formation. Calcein fluorescence indicates cell viability and activity of endogenous esterases. Scale bar: 10 µM. (B) Calcein fluorescence correlates with plating density of cells per well, at 5 days in vitro. Data is shown as mean ± s.e.m. (n=3). (C) tBHP treatment reduces cell viability, which was confirmed by MTT assay. Data is shown as mean ± s.e.m. (n=3). (D) Representative neuroprotection experiment utilizing 3-NP as insult and 1 mM Trolox™ as neuroprotectant. Trolox™ pre-treatment prevents 3-NP-induced loss in cell viability. Data is shown as mean ± s.d.

Figure 2. Ca²⁺ mobilization assays in 96-well plate format using primary cultured cortical neurons

(A) Depolarization of primary cultured neurons with 100 mM KCl (stimulus indicated by arrow) increases the intracellular Ca²⁺ concentration 4.45-fold. Data from three separate neuronal cultures is shown as mean ± s.e.m. and as ratio (F/F₀) of the relative fluorescence (F) and normalized background fluorescence (F₀). (B) Absolute RFU values obtained for the experiment shown in (A) were 33 ± 3 for background and 145 ± 17 for the maximal response (n=3, P<0.01). Data is shown as mean ± s.e.m. ** P<0.01. (C) Representative experiment showing the effect of the Ca²⁺ channel blocker nifedipine on the intracellular Ca²⁺ concentration, to highlight experimental variability. Data is shown as mean ± s.d. The arrow indicated KCl stimulus. (D) Nifedipine reduces the maximal response (F/F₀ max) by 38%. Data is shown as mean ± s.d.