Gexscope tissue preservation solution

The bladder samples employed in this study were procured from patients who underwent transurethral resection of bladder tumors at Shenzhen Luohu District People's Hospital, in accordance with the guidelines established by the Ethics Committee Board of the aforementioned hospital (2020‐LHORMYY‐KYLL‐007). Written informed consent was obtained from all participants. After surgery, fresh tissue samples were collected and preserved in the GEXSCOPETM Tissue Preservation Solution (Singleron, Cologne, Germany) for subsequent processing. Tissue samples were initially washed with phosphate‐buffered saline (PBS; Gibco, USA), then cut into small pieces of 1–2 mm, and digested using GEXSCOPETM Tissue Dissociation Solution (Singleron) at 37°C with oscillation for 15 min. Following digestion, the samples were passed through a 40‐µm sterile strainer, and the supernatant was removed by centrifugation at 1000 rpm for 5 min at 4°C. The cell pellet was then resuspended in 1 mL PBS, treated with 2 mL GEXSCOPETM red blood cell lysis buffer (Singleron), and incubated on ice for 10 min to lyse the red blood cells. Lastly, the single‐cell suspension was collected, resuspended in PBS, and subjected to further analysis.

Sample collection was carried out under license in accordance with the Guidelines for Care and Use of Laboratory Animals of China, and all protocols were approved by the Institutional Review Board of Nantong University. Five 42-day-old (P42) and ten 56-day-old (P56) C57BL/6J mice were used in this study. After sacrificing the mice, the epididymis was dissected and divided into three regions (caput, corpus, and cauda) as previously described^{7 (link),74 (link)}. Briefly, the mice were sacrificed by anesthetized cervical dislocation, and the epididymis was quickly isolated and placed in a petri dish with ice-cold saline. Epididymal fat was carefully removed, and the sample was separated into caput, corpus, and cauda regions under a dissecting microscope. The different segments of each epididymis were immediately transferred into GEXSCOPE^TM Tissue Preservation Solution (Singleron Biotechnologies) in Eppendorf tubes labeled EP (caput), EO (Corpus), and ED (cauda) on ice. The procedure was repeated in five mice until all segments were collected in each tube. Two replicates for each pooled segment of the P56 epididymis were prepared. The pooled segments were further processed following the procedure of tissue dissociation and single-cell preparation.

Two groups [Sb, 0 mg/mL (Control) and Sb, 1.2 mg/mL (Sb)] were chosen for scRNA-seq based on the above results. For each group, the testes of 180 male flies were dissected in cold PBS. The resulting mixtures of testes were washed with cold PBS three times and immediately transferred into GEXSCOPETM Tissue Preservation Solution (Singleron Biotechnologies, Cheshire, CT, USA) on ice. The pooled segments were further processed following the procedure for tissue dissociation and single-cell preparation.