Fitc nhs

Poly‐d‐Lysine (PDL) (P0899), DNase 1 type IV, Triton X‐100, FITC‐NHS, Rhodamine 6G (Rh6G), fluorescein sodium salt (FITC), and 1,1′‐Dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate were purchased from Sigma‐Aldrich. Alexa‐Fluor 488 goat anti‐mouse, 4′,6‐diamidino‐2‐phenylindole, paraformaldehyde (PFA, 7 230 681), goat serum, Dulbecco's modified eagle medium (DMEM), penicillin/streptomycin (Pen/Strep), B27 supplement (17 504 001), GlutaMAX, neurobasal medium, Fluoromount‐GTM mounting medium (00‐4958‐02), fetal bovine serum (FBS), CMFDA, and YO‐PRO‐1 iodide were purchased from Life Technologies. Mouse anti‐Tuj1 antibody (801 202) was purchased from Biolegend. Rabbit anti‐GFAP (Z0334) was obtained from DAKO. Papain suspension was purchased from Worthington. Cell strainer (70 µm) was obtained from BD, Biosciences, USA.

To determine the surface density of bound glycans, an indirect approach was necessary. PMMA plates (25 × 60 mm) were activated with the amino-linker hexamethylene diamine as described above. Then an aminoreactive fluorescein isothiocyanate (FITC-NHS, Sigma) at a concentration of 100 µM ml⁻¹ was coupled according to the protocol for glycan bonding. The unspecific adsorbtion of the FITC was determined by incubation of PMMA plates which were treated according to the protocol but lacking the hexamethylene diamine. PMMA plates initially coated with sandfish glycans were subsequently incubated with the FITC-NHS. The number of bound FITC molecules was determined by the reduction of fluorescence of the supernatant of the individual incubations. Fluorescence was determined using a plate reader (Glomax, Promega, Germany) with the FITC/Cy2 filter block.