V900893

Single testicular cells were isolated using a two-step enzymatic digestion protocol described previously (Sohni et al., 2019 (link)). Testes from P20 WT or SCARKO male mice were dissected and decapsulated in PBS. Collagenase Type IV (Sigma-Aldrich, V900893, final concentration of 1.0 mg/mL) was added to the tubules and incubated at 37°C for 5 min with gentle agitation. The separated tubules were washed with PBS twice and followed by digestion with trypsin (Gibco, 15090046, final concentration of 0.6 mg/mL) and DNase I (Sigma-Aldrich, DN25, 10 ku/mL) at 37°C for 20 min with periodic vigorous agitation. The cells were filtered through a 40 μm cell strainer and pelleted by centrifugation at 600 g for 5 min at 4°C. After washing with PBS twice, dissociated cells were used for scRNA-seq.

The experimental ICH model was generated as described in our previous study (Liu et al., 2016 (link)). Briefly, rats were anesthetized using sodium pentobarbital (55 mg/kg) and then fixed in the stereotactic frame facing downward. During the surgical procedure, the rectal temperature of the rats was maintained at 37 ± 0.5°C using a rectal thermostat probe and heating pad. In addition, the heart rate and arterial blood pressure of the rats were monitored using a Data Acquisition Systems (AD Instruments, Milford, MA, United States). A burr hole with a diameter of 3 mm was carefully drilled using an electric drill along the right coronal suture at 3.0 mm lateral and 0.5 mm posterior to the bregma. A 30-gauge (G) needle was inserted into the right caudatum with its tip 6 mm beneath the dural surface. Afterward, 0.3U collagenase IV (Sigma-Aldrich V900893, United States) in 1.0 μl saline was slowly injected into the right caudatum for 10 min and the needle removed from the caudatum 20 min after injection. The burr hole was sealed with bone wax and the incision on the skin was sutured. For rats in the CLB + ICH group, the ICH model was generated 24 h after generation of the CLB model.

FACS analysis was performed to analyze immune cell populations of innate immunity and adaptive immunity in inflammatory skin. Briefly, skin tissues were digested with collagenase D (V900893, Sigma) and Dnase 1 (D8071, Solarbio, Beijing, China) to prepare a single cell suspension. The cell suspension was then stained with zombie violet viability dye, and blocked non-specific Fc-mediated interactions with CD16/CD32 Monoclonal Antibody. Then, cells were incubated with the indicated antibody cocktail mix (Panel A or B, listed in Table 1) for 1 h at 4 °C with shaken every 10 min gently. Finally, the cells were resuspended in stabilizing fixative buffer (#338036, BD biosciences, San Jose, CA, USA). FACS analysis was performed using the Thermo Attune NxT machine (Waltham, MA, USA) and further analyzed using FlowJo V10 software.