Large cell column

Tissue samples of the skin were obtained from the neck of 9 dogs before and after HSCT on days +28, +56 and +105 under general anaesthesia (punch biopsies, 2 × 50.5 mm²). In long-term chimeras specimen of dermal tissue were also taken after day +105. Tissue samples were disinfected in povidone-iodine (Mundipharma, Limburg/Lahn, Germany), bleached with sodium thiosulfate (0.05 %, Sigma Aldrich, Hamburg, Germany) and washed in phosphate buffered saline (PBS, Biochrom AG, Berlin, Germany). The epidermis was separated from the dermis by digestion with dispase (2.24 U/ml, Roche, Mannheim, Germany) at 4 °C overnight and at 37 °C (water bath) for one additional hour. Subsequently the epidermis was incubated at 37 °C for 30 min in trypsin (0.25 %, Biochrom AG) with DNase (10 μl/ml, Roche) to obtain a single cell suspension.
Single cells were labelled with a monoclonal mouse anti-canine CD1a antibody (clone CA9.AG5; kindly provided by Dr. P.F. Moore, School of Veterinary Medicine, University of California). Afterwards cell suspension was incubated with a goat-anti-mouse MicroBead (Miltenyi Biotec, Bergisch Gladbach, Germany). The labelled LC were enriched by MiniMACS device using large cell columns (Miltenyi Biotec).

As previously reported in detail by [19 (link)], retinal cell types were isolated from murine retinas and sorted into cell fractions with the Miltenyi magnetic cell sorting system. Retinas were immediately removed from enucleated eyes and digested in 12 mM PBS glucose containing 0.2 mg/ml papain (Roche). For 30 min 37 °C retinas were washed and treated with Dnase I in PBS/Glucose (200 U/ml, 4 min, room temperature). Supernatant was replaced with an extracellular solution (136 mM NaCl, 3 mM KCl, 10 mM HEPES, 11 mM glucose, 1 mM MgCl₂ and 2 mM CaCl₂, pH 7.4) and the tissue dissociated. Cell types were subsequently subtracted from the cell suspension by incubating it with specific antibodies coupled to magnetic microbeads (CD11b for microglia, CD31 for perivascular cells and CD29 for Müller cells (Milteny Biotec)). In brief, for every purification step, the suspension was pipetted onto and large cell column (Milteny Biotec, Cat. 130-042-202), clipped to a magnetic rack. Non-magnetically labeled cells were eluted by washing. The column was removed from the rack and labeled cells were eluted, spun down (10,000 × g, 15 min, 4 °C) and the pellet was immediately frozen in dry ice. Finally, the neuron rich cell suspension depleted of microglia, vascular and Müller cells was collected in the same manner.