Optimised techniques for high-throughput screening of differentiated SH-SY5Y cells and application for neurite outgrowth assays

Abstract

Neuronal models are a crucial tool in neuroscientific research, helping to elucidate the molecular and cellular processes involved in disorders of the nervous system. Adapting these models to a high-throughput format enables simultaneous screening of multiple agents within a single assay. SH-SY5Y cells have been widely used as a neuronal model, yet commonly in an undifferentiated state that is not representative of mature neurons. Differentiation of the SH-SY5Y cells is a necessary step to obtain cells that express mature neuronal markers. Despite this understanding, the absence of a standardised protocol has limited the use of differentiated SH-SY5Y cells in high-throughput assay formats. Here, we describe techniques to differentiate and re-plate SH-SY5Y cells within a 96-well plate for high-throughput screening. SH-SY5Y cells seeded at an initial density of 2,500 cells/well in a 96-well plate provide sufficient space for neurites to extend, without impacting cell viability. Room temperature pre-incubation for 1 h improved the plating homogeneity within the well and the ability to analyse neurites. We then demonstrated the efficacy of our techniques by optimising it further for neurite outgrowth analysis. The presented methods achieve homogenously distributed differentiated SH-SY5Y cells, useful for researchers using these cells in high-throughput screening assays.

Human repair‐related Schwann cells adopt functions of antigen‐presenting cells in vitro

“…The phagocytic capacity of hrSCs toward cell‐extrinsic material prompted us to evaluate whether pathways associated with inflammation and antigen presentation were active in hrSCs. To this end, we interrogated the transcriptome data generated by deep RNA‐sequencing (RNA‐seq) of hrSC cultures ( n = 5) and of neuroblastic tumor cells (NB cells) ( n = 5), which represent an established model for neuronal cells broadly used in neuroscience as shown by us and others (Attoff et al, 2020 ; Dravid et al, 2021 ; Schikora et al, 2021 ; Taylor‐Whiteley et al, 2019 ; Wang et al, 2016 ; Weiss et al, 2016 , 2021 ). We determined the differentially expressed genes in neuronal NB cells versus repair‐like SCs to obtain GO terms enriched in the latter as basis for further analysis.…”

“…The phagocytic capacity of hrSCs toward cell-extrinsic material prompted us to evaluate whether pathways associated with inflamma- others (Attoff et al, 2020;Dravid et al, 2021;Schikora et al, 2021;Taylor-Whiteley et al, 2019;Wang et al, 2016;Weiss et al, 2016Weiss et al, , 2021. We determined the differentially expressed genes in neuronal NB cells versus repair-like SCs to obtain GO terms enriched in the latter as basis for further analysis.…”

Balamuthia mandrillaris trophozoites ingest human neuronal cells via a trogocytosis-independent mechanism

“…Thus, SH-SY5Y is more relevant to human physiology than other cell lines. Notably, human SH-SY5Y cells can be induced to differentiate into mature neurons [ 27 ], allowing the screening of cytotoxicity in a high-throughput manner with more physiological relevance than undifferentiated cells [ 28 ].…”

Neuroprotective effect of phospholipase A2 from Malaysian Naja sumatrana venom against H2O2-induced cell damage and apoptosis

“…Treatment with retinoic acid resulted in cells that acquired neuron-like phenotype increased homogenous cell population and expressed tyrosine kinase receptor B (TrkB) on the surface of the cells after 3 days of incubation (Encinas et al, 2000). The introduction of BDNF, an agonist of the TrKB receptor, continues to expand the neurite length of cells, increase d the connection between cells, and thus, increase the cells survival (Encinas et al, 2000;Dravid et al, 2021). Other than RA and BDNF, we added dcAMP, EGF and FGF to maintain the cell growth and neurite outgrowth of SH-SY5Y to mature neuron cells (Even et al, 2009;Pirou et al, 2017;Huang et al, 2021) (Figures 1B-D).…”