Papain and collagenase dispase

Trigeminal ganglia (TG) were isolated from thy1-YFP (yellow fluorescent protein) transgenic mice in which the nerves show yellow fluorescence and cultured as described previously [12 (link), 14 (link)]. In brief, ophthalmic branches of the trigeminal nerves were harvested and cleaned from contaminating tissue, then subjected to enzymatic digestion with papain and collagenase/dispase (Worthington Biochemicals, Lakewood, NJ) and separated in Percoll gradients by centrifugation. Cell cultures were maintained in 35 mm glass-bottom dishes coated with 20 μg/mL laminin/poly-D-Lysine (Sigma, St. Louis, MO) and supported with media (Neurobasal A medium supplemented with 1% B27; Invitrogen, Carlsbad, CA) supplemented with 2% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (ABAM; Gibco, Grand Island, NY).

Trigeminal ganglia (TG) were removed from thy1-YFP mice and cultured as previously described [20 (link), 23 (link)]. In brief, ophthalmic branches of the trigeminal nerves were harvested and cleaned from contaminating tissue, then subjected to enzymatic digestion with papain and collagenase/dispase (Worthington Biochemicals, Lakewood, NJ) and separated in Percoll gradients by centrifugation. Cell cultures were maintained in 96-well plates coated with 20 μg/mL laminin (Sigma, St. Louis, MO) and supported with media (Neurobasal; Invitrogen, Carlsbad, CA) supplemented with 2% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (ABAM; Gibco, Grand Island, NY). Mouse corneal epithelial sheets were isolated using dispase treatment, as described by Kawakita et al.[24 (link)]. Epithelial sheets were rendered into single cells by 0.25% trypsin-EDTA (Gibco). Dissociated cells were seeded in six-well plates with corneal epithelium growth medium (Epilife; Cascade Biologics, Portland OR) with 20 μM calcium, 1% human corneal growth supplement (Cascade Biologics), 0.01 μg/mL mouse epidermal growth factor (Sigma), 0.1 nM cholera toxin A subunit from Vibrio cholerae (Sigma), and 1% ABAM (Gibco) until they were 90% confluent.