India ink

GFP-expressing SKPs were liberated from differentiation culture and suspended in NeuroCult® NS-A Basal Medium (Rat) with 10% Differentiation Supplement (StemCell Technologies) (StemCell Technologies) with 15% v/v pH-neutralized rat tail collagen^{17 (link)} in differentiation media and 2% v/v India ink (Becton, Dickinson and Company, Franklin Lakes, NJ) at a density of 5 × 10⁵ cells/mL. SKPs were injected 24–46 days after initial plating. Adult recipient rats (n=7) underwent a second laparotomy under general inhaled anesthesia between 65–85 days after segmental jejunal denervation. The denervated segment was identified and SKPs were injected subserosally using a micro-injector as described previously.²⁷ Injected segments were covered with omentum, and the abdomen and skin closed. After 1, 2, 7, 14, 21, and 28 days, the animals were euthanized and the experimental segments fixed for IHC evaluation.

C. neoformans H99 was cultured overnight in YPD at 30 °C, and then, the cells were washed twice in sterile PBS and diluted to 1 × 10⁶ CFU/ml in Dulbecco’s Modified Eagle medium (DMEM; Gibco 11965–092) or 10% Sabouraud dextrose broth pH 8. DMSO or indicated concentrations of drug was added to cells, and then cells were dispensed into 24-well plates and incubated at 37 °C for 24 (10% Sab) or 48 h (DMEM, with 5% CO₂). Cells were washed in PBS, then mixed in a 1:1 ratio with India ink (Becton, Dickinson and Company) on a microscope slide. Brightfield images were acquired on a Nikon epifluorescence microscope with a Cool Snap HQ2 camera and Nikon Elements image acquisition and analysis software. For cell separation quantification, cells grown under 10% Sab conditions for 24 h with 1 μg/mL 1 were washed in PBS, and then brightfield images were acquired. Cell bodies were defined rounded cellular compartments separated by invaginations. At least 150 cells were counted per sample of triplicates.

pH-neutralized rat tail collagen was prepared using our laboratory’s previously-described protocol.^{11 (link)} SKPs were liberated and suspended in differentiation media with 15% v/v neutralized rat tail collagen in differentiation media and 2% v/v India ink (Becton, Dickinson and Company, Franklin Lakes, NJ) at a density of 5 × 10⁵ cells/mL. SKPs were transplanted at days 24, 41, or 94 after initial plating. Recipient rats (n=9) underwent a second laparotomy under isoflurane anesthesia at post-BAC treatment day 21–79. The isolated BAC-treated segments were identified and meticulous adhesiolysis was performed. The SKP suspension was injected subserosally in a fanning linear fashion using a micro-injector with a 0.5-inch long 33-gauge needle (Hamilton, Reno, NV). Approximately 100–200 μL of suspended SKPs were injected into each segment. After injection, the isolated segment was wrapped with mobilized omentum, replaced into the peritoneal cavity, and the abdomen and skin were closed. At 1, 6, or 9 days post-transplant, the animals were euthanized and the injected segments collected and fixed for immunohistochemistry (IHC). Recipient muscularis thickness and the presence of native ganglia were compared among segments injected at earlier versus later time points after BAC treatment.