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

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.

Optimization of estrogen receptor analysis by immunocytochemistry in random periareolar fine-needle aspiration samples of breast tissue processed as thin-layer preparations.

Immunostaining of estrogen receptor alpha (ER) in samples of benign breast tissue obtained by random periareolar fine-needle aspiration (RPFNA) is a practical tool for breast cancer chemoprevention trials. The authors report an optimized method of ER immunostaining for use with thin-layer preparations of modified Cytolyt-fixed benign breast tissue acquired by RPFNA. Samples of benign breast tissue and MCF-7 controls processed as thin-layer preparations were tested for the effects of antibody titer, antigen retrieval temperature (90 degrees or 115 degrees C), buffer (20% nuclear decloaker, pH 9.3; 10 mM citrate buffer, pH 6), and blocking solution (0.01% glucose oxidase or 0.3% H2O2) on ER immunostaining. The prevalence of positively stained breast epithelial cells, mean intensity of ER staining, and composite immunostaining score were evaluated for effect of immunostaining protocol. RPFNA samples and MCF-7 cells processed using nuclear decloaker and low-temperature antigen retrieval had more ER-positive cells (P<0.0001) and increased mean staining intensity and weighted staining indices (P<0.05) compared with samples prepared with citrate buffer and high-temperature antigen retrieval. Glucose oxidase increased ER-positive cells in comparison to hydrogen peroxide (P<0.04) when combined with low-temperature antigen retrieval and the use of nuclear decloaker. Staining was negative for all non-immune controls regardless of protocol. The combination of low-temperature antigen retrieval, diluted commercial nuclear decloaker solution, and a glucose oxidase blocking step yielded optimal ER immunostaining for thin-layer preparations of benign breast tissue harvested by RPFNA.

Comparison of cytomorphology in specimens obtained by random periareolar fine needle aspiration and ductal lavage from women at high risk for development of breast cancer.

Ductal lavage (DL) and random periareolar fine needle aspiration (RPFNA) are both being used to harvest epithelial cells for risk assessment as well as response evaluation in chemoprevention trials. The magnitude of increase in relative risk has been defined in a prospective study for RPFNA but not for DL atypia. We attempted both procedures in 26 women at high risk for development of breast cancer. Median age was 43 (range 32-57); 15 women were premenopausal, with 6 of the postmenopausal women on HRT. Collection of nipple aspirate fluid (NAF) was attempted and, if successful, was followed by DL; RPFNA was then performed on all women. Both procedures were attempted the same day (follicular phase of menstrual cycle if premenopausal) in 24 subjects and within three months for two subjects. Twenty-three subjects produced NAF, 17 of the 23 (74%) had a successful duct cannulation as part of the DL procedure, with 16 yielding sufficient (10) ductal cells for morphologic assessment. Twenty-five of 26 (96%) subjects had a successful RPFNA procedure with adequate cellularity for morphology. There was concordance between DL and RPFNA specimens for traditional cytologic category assessment in 10/16 (63%), Masood index score in 13/16 (82%), and Consensus Panel assessment in 12/16 (75%) of specimens. We conclude that same day DL and RPFNA is feasible, with 62% and 96% of high-risk women having a successful procedure with evaluable cytomorphology. RPFNA was more likely to yield an evaluable specimen, but if a cellular DL specimen was obtained, morphology was generally similar.