To isolate and culture wild-type and Pcdhg mutant SACs in vitro, we crossed the Thy1-OFP3 transgene, which selectively directs expression of Kusabira Orange (OFP) in SACs and subset of RGCs37, into Pcdhgfcon3; Six-cre mice. Retinas from genotyped Pcdhgfcon3/fcon3; Six-cre Thy1-OFP3 mutant and control P2 mice were dissociated using papain22. OFP+ SACs were isolated by fluorescence activated cell sorting (FACS, MoFlo), plated onto poly-L-lysine-coated glass coverslips (Warner) and cultured 7-9 days in RGC growth media modified from Meyer-Franke et al.43 in the following ways: (a) substitution of NS2144 for B27, (b) substitution of N2 (Invitrogen) for Sato stock, (c) addition of TGF-β1 and -β2 (2.5ng/mL; Peprotech), and (d) addition of mouse glia-conditioned medium (15%). One-third of media was exchanged with fresh media every three days. Cells were fixed with cold 4%PFA/4% Sucrose for 15min, and immunostained for syntaxin and calbindin to confirm SAC identity, and for GFP to confirm Pcdhg−/−; GFP− SACs from unrecombined Pcdhg-GFP+ SACs due to variegated Six3-Cre activity in retina.
