Abstract
Preclinical studies have suggested that increased adult neurogenesis in the hippocampus might have potential therapeutic effects for Alzheimer's disease and depression; therefore, it is a target for the treatment of some brain diseases. In this technical communication, we propose a cell-based fluorescence assay to study the neurogenesis of adult hippocampal progenitor cells that can be used for high-throughput screening of drugs promoting neurogenesis. Three fluorescent dyes (DAPI, Alexa Fluor 488, and Alexa Fluor 594) and a fluorescence spectrophotometry reader were used, which confirmed that the mutual interference of the three fluorescent dyes is very low. We used this cell-based fluorescence assay to evaluate the effects of three neurotrophic factors, ciliary neurotrophic factor (CNTF), insulin-like growth factor 1 (IGF-1), and IGF-2 on the promotion of neurogenesis in adult hippocampal neural progenitor cells. The fluorescence intensity ratio of the neuronal marker, class III β-tubulin, to the housekeeping protein, glyceraldehyde 3-phosphate dehydrogenase, or nuclear staining dye, DAPI, in CNTF-treated cells was significantly higher than in control cells. The ratios in IGF-1 and IGF-2-treated cells were slightly higher under higher cell density conditions. These results are consistent with those in previous reports; therefore, this report proved the efficacy of this method. Taken together, the results showed that this simple, rapid, and economical cell-based immunofluorescence assay could be a powerful tool for the rapid screening of drugs that promote adult neurogenesis.
Highlights
Preclinical studies have suggested that increased adult neurogenesis in the hippocampus might have potential therapeutic effects for Alzheimer’s disease and depression; it is a target for the treatment of some brain diseases
The results showed that for a single fluorescent dye sample, the fluorescence intensity (FI) value is very low if read at a non‐corresponding filter setting and the differences in FI value in the mixture of fluorescent dyes compared to a single fluorescent dye were less than 7%
The experimental results are consistent with the analysis results from the Fluo‐ rescence Spectra Viewer; that is, the mutual interfer‐ ence of the three fluorescent dyes is very low (Tables I, II, and III). We used this cell‐based fluorescence assay to eval‐ uate the effects of three neurotrophic factors on pro‐ moting neurogenesis of adult hippocampal neural progenitor cells; ciliary neurotrophic factor (CNTF) significantly induced neuronal differentiation, while
Summary
Kun Zhang1†, Bin Li2,3†, Peifang Li1, Xiaoli Yang, Huixian Cui and Xiaoyun Liu1,5*. We propose a cell‐based fluorescence assay to study neurogenesis in adult hippocampal progenitor cells This method is advantageous because it uses 96‐well fluorescence plate readers, which enables a large amount of experimental data, and dozens of compounds, to be read simultane‐ ously. The experimental results are consistent with the analysis results from the Fluo‐ rescence Spectra Viewer; that is, the mutual interfer‐ ence of the three fluorescent dyes is very low (Tables I, II, and III) We used this cell‐based fluorescence assay to eval‐ uate the effects of three neurotrophic factors on pro‐ moting neurogenesis of adult hippocampal neural progenitor cells; ciliary neurotrophic factor (CNTF) significantly induced neuronal differentiation, while
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