DEPARTAMENTO DE CIÊNCIAS DA VIDA ... - Estudo Geral

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II - Materials and methods
II.1 - Materials
II.1.1 - Cell culture
Cell culture dishes were from Corning-Costar. Cell culture Neurobasal ○ R
medium (1x),
MEM-Alpha Medium (1x) and Dubecco’s Phosphate Buffered Saline 10x were from
Gibco ○ R
. 0,05% Trypsin-EDTA (1x), Penicillin(10000 units/ml) / Streptomycin (10mg/ml)
solution and Fungirone ○ R
Amphotericin B (250 µg/ml) were from Invitrogen. MSCqualified
Fetal Bovine Serum (FBS) was from Hyclone - Thermo. Medium supplement
B27 was from Invitrogen and human recombinant EGF and bFGF were from Peprotech.
BSA was from Calbiochem and proteins used to promote cell attachment Collagen
type-I (COL-1) from rat tail and human Fibronectin (FN) isolated from plasma were
from BD Bioscience and Roche, respectively. All Biological processing was done under
sterile conditions using a class-II biosafety vertical air flow cabinet (HeraSafe HS-18,
Heraeus). The centrifugation was done using a centrifuge 5810 R from eppendorf. Cells
were observed with an Axiovert 40C ZEISS microscope and maintained in a Shel LAB
CO 2 Series incubator.
II.1.2 – Polyacrylamide hydrogels
Acrylamide and bis-acrylamide were purchased from Bio-Rad and Applied Chem,
respectively. Ammonium persulfate (APS) and dichlorodimethylsilane,
Tetramethylethylenediamine (TEMED), NHS (N-Acryloxysuccinimide) and 3-
(trimethoxysilyl) propyl methacrylate were from Sigma-Aldrich, Fluka and Santa Cruz
Biotechnology, respectively. The gel polymerization system was Mini protean-3 from
Bio-Rad.
II.1.3 - Immunocytochemistry
Alexa 488-conjugated donkey anti-mouse and Alexa 568-conjugated goat anti-rabbit
antibodies were purchased from Invitrogen. Anti-O4 and anti-Nestin mouse antibodies
and anti-Beta-III-Tubulin and anti-GFAP rabbit antibodies are from R&D, Millipore,