Hank s medium

After an ovariohysterectomy on a healthy 1-year-old dog, a biopsy from the uterine endometrium was performed to isolate C-EnSCs. The sample was placed in a tube containing Hank’s medium (Gibco, Waltham, MA, USA) containing 5% penicillin-streptomycin (Gibco, USA). The tissue sample was cut into small pieces after several washing cycles. The crushed pieces were added to a falcon plastic tube containing type 1 collagen enzyme (Sigma, Ronkonkoma, NY, USA) at a 2 mg/mL concentration and incubated for two hours at 37 °C in a CO₂ incubator (Sina, Hombarat, Iran). In the next step, DMEM/F12 (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12; Gibco, USA) with 10% fetal bovine serum (FBS; Gibco, USA) was added to each sample and pipetted. To remove undigested tissues, the resulting solution was passed through a 70 μm mesh once and a 40 μm mesh twice. Blood cells were also removed with Ficoll (Sigma, USA). Isolated cells were transferred into DMEM/F12 medium supplemented with 15% FBS and streptomycin-penicillin 1%. Then, three passages were performed with Trypsin-EDTA (Gibco, USA) [16 (link)].

Whole blood obtained was collected in EDTA-buffer tubes. Samples were subjected to red-blood-cell lysis for further analysis using flow cytometry. Bone marrow cells were harvested by flushing femurs with Hank’s Medium (Hanks’ Balanced Salt Solution + 0.3 mmol/l EDTA + 0.1% BSA) (Gibco by life technologies). Spleen and lymph nodes were mechanically crushed and passed through a 30 μm cell strainer (Cell-Trics, Partec) using Hank’s Medium and to obtain single cell suspensions. Single cell suspensions were subsequently stained with different antibody cocktails and analyzed using a FACS Canto II, using the FACSDiva software (BD Biosciences). Cell populations were discriminated by the following antibody cocktail: anti-CD45, anti-CD115, anti-Gr1, anti-CD11b, anti-B220, anti-CD3, anti-CD4, anti-CD8 (All eBioscience). Cell populations and marker expression were gated as depicted below using the FlowJo analysis program (Treestar): neutrophils (CD45⁺CD115⁻Gr1^high), classical monocytes (CD45⁺CD115⁺Gr1^high), non-classical monocytes (CD45⁺CD115⁺Gr1^low), B-cells (CD45⁺CD115⁻Gr1⁻B220⁺), CD3⁺T-cells (CD45⁺CD115⁻Gr1⁻CD3⁺), CD4⁺T-cells (CD45⁺CD115⁻Gr1⁻CD3⁺CD4⁺) and CD8⁺T-cells (CD45⁺CD115⁻Gr1⁻CD3⁺CD8⁺).