Liberase dh

All animal experiments were carried out according to procedures approved by The Jackson Laboratory Animal Care and Use Committee (ACUC). The following mouse stocks were obtained from The Jackson Laboratory: (B6)C57BL/6J-#000664, (NSG)#005557, (Ncr1^gfp), #022739, (Prf1 knockout [KO])#022739, (Klrk1⁻) # 022733, and (UBC-GFP) # 004353. Ncr1^{tm1.1(icre)Viv} (MGI:5308410) mice were a kind gift from Eric Vivier and Stephen Waggoner. NKP46 DTR mice were generated by intercrossing Ncr1^{tm1.1(icre)Viv} (MGI:5308410) with ROSA^DTR (MGI:3772576) mice. P3 amputations performed on age-matched 8- to 10-week-old male mice anesthetized with isoflurane before amputation of the distal third of the terminal phalanges (Storer and Miller, 2020 (link)). Two digits per hindpaw were amputated, with the middle digit used as an uninjured control (Figure 1A). Buprenorphine was given at 1 mg/kg via a subcutaneous injection. After amputation, the digits were sprayed with Cavilon No-Sting Barrier Film (3M) to minimize potential infection. For NKP46⁺ cell depletion in NKP46 DTR mice, 200 ng DT was injected intravenously (i.v.) at 3-day intervals. Blastemal cells were isolated via mechanical dissociation on glass dishes (MatTek, P35G-0-10-C) and then digested with 1 U/mL Liberase-DH (Roche, 5401054001) in tubes for 60 min at 37°C. Dissociated cells were used for FC and cytotoxicity assays.

Mouse ILCs were prepared as described [14 (link)]. Briefly, the small intestines were extracted from C57BL/6 mice. After removal of the surrounding mesentery and Peyer’s patches, and fecal matters, the small intestines were minced into longitudinal pieces of 7 cm, and further cut into 1 cm pieces, followed by vigorous washes in phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10.1 mM Na₂HPO₄, and 2 mM KH₂PO₄, pH 7.4). They were further treated with 1 mM EDTA/PBS at 37°C for 20 min for removal of the intraepithelial lymphocyte and intestinal epithelial cell fractions. Thereafter, the samples were harvested by centrifuging for 20 min at 500×g, followed by vigorous washing for 20 sec in PBS. Further, they were minced into 1–2 mm pieces, and incubated in 10 ml of pre-warmed RPMI medium supplemented with 2% fetal bovine serum containing 20 μg/ml Liberase DH (Roche, Basel, Switzerland) and 50 μg/ml DNase I (Roche) at 37°C for 30 min. The cells were subsequently passed through a 70 μm EASY strainer (BD Biosciences, San Jose, California, USA), and a 37 μm nylon filter (BD Biosciences). The flow-through cells were harvested by centrifuging for 5 min at 500×g, 4°C, and then used as an ILC fraction.