Neuronal Cell Culture Protocol

Hanks' balanced salt solution (HBSS), serum-free neurobasal media (NB), B27 supplement (50x), L-glutamine, Prolong Gold anti-fade reagent with 4′,6-diamidino-2-phenylindole (DAPI), SuperScript III reverse transcriptase, SYBR/Green Master mix, and Alexa 405-, 488-, or 555-conjugated secondary antibodies were obtained from ThermoFisher. Poly-L-lysine hydrobromide, cytosine β-D-arabinofuranoside hydrochloride (Ara-C), and anti-actin antibody were from Sigma. Microtubule-associated protein 2 (MAP-2) antibody was obtained from Abcam. C5aR1 antibody (10/92) was obtained from SeroTec (Ager et al., 2010 (link)). Anti VGLUT1 and anti GAD67 antibodies were obtained from Millipore. HRP-conjugated F(ab')₂ antibodies were from Jackson ImmunoResearch Laboratories. Human C5a peptide was obtained from CompTech. PMX53 was obtained from Cephalon. All buffers are made with Millipore-purified water with additional filters to eliminate LPS and are periodically tested for endotoxin using the Limulus amebocyte lysate clot assay (all solutions added to cells were <0.1 EU/mL; 1 EU is equivalent to 0.1 ng/mL LPS).