Spectramax paradigm multi mode microplate detection reader

To assess adhesion of mouse BMDMs, 96-well plates coated with 2.5 μg type 1 collagen (from rat tail, Sigma) per well and blocked with PBS containing 0.5% BSA were used. Assays were performed by plating 5 × 10⁴ CFDA-SE-labeled (Molecular Probes) BMDMs per well. After 30 min incubation, non-adherent cells were removed by washing with PBS and fluorescence emitted by adhering cells was measured (excitation: 492 nm; emission 517 nm) using a SpectraMax Paradigm Multi-Mode Microplate Detection reader (Molecular Devices).

Microdissection of the CGE was performed at E14.5, trypsinized dissociated cells were plated on 96-well plates and cultured in NMB supplemented with mCPBG (10 μM; Tocris) and the mitotic inhibitor arabinofuranosyl cytidine (1 μM). Quantification of protein levels was performed using ELISA kit (Thermo scientific) and 5-HT_3AR antibody (1:1,000, LSBio) on E14.5 day in vitro 1 (+DIV1) or E14.5 (+DIV4) cells. Absorbance levels were measured on a SpectraMax Paradigm Multimode Microplate detection reader (Molecular Devices). Absorbance levels for the 5HT_3AR were normalized to absorbance levels calculated for the total amount of cells via Janus Green whole-cell staining.