Rbc lysis solution

High molecular weight DNA was extracted both from fresh (<5 days old) and frozen (– 80 °C) whole blood. DNA extraction was performed following the manufacturer’s guidelines (PlugLysis, Bionano Genomics, USA). RBC lysis solution (Qiagen) was used to lyse red blood cells and pellet white blood cells. The white blood cells were re-suspended in cell suspension buffer (Bio-Rad) and embedded into agarose plugs (CHEF Genomic DNA Plug Kit, Bio-Rad) to lessen fragmentation of long DNA molecules during the overnight lysis at 50 °C using a 16:1 ratio of lysis buffer (Bionano Genomics, USA) and Puregene Proteinase K (Qiagen). The plugs were washed with Tris-EDTA buffer and digested at 43 °C with GELase (Epicentre). Extracted high molecular weight DNA was purified from digested materials/enzymes via drop dialysis using Millipore membrane filters (EMD Millipore, USA) placed on Tris-EDTA buffer. DNA quantifications were carried out using Qubit dsDNA assay kits with a Qubit 3.0 Fluorometer (ThermoFisher Scientific).

All samples were processed within 12 h from collection. Whole blood was lysed prior to staining using the RBC Lysis solution (Qiagen, Germany) according to manufacturer's specification. Immune populations were identified by staining with fluorophore-conjugated anti-CD11b, anti-CD33, anti-CD3, anti-HLA-DR, anti-CD45, anti-CD68, anti-CD206, anti-CD163, (BioLegend), anti-CD14, anti-CD15 (eBioscience) antibodies on ice for 30 min. Fluorescence data was acquired using BD Accuri C6 (BD Biosciences), Cyan and/or CytoFLEX (Beckman Coulter) cytometers. Normalised population statistics –including the median fluorescence intensities (MFI) were determined using FlowJo (BD Biosciences, formerly developed by FlowJo LLC).
Where indicated cell death was assessed by propidium iodide staining of cells after 72 h incubation with Gemtuzumab ozogamicin (Gift from Pfizer).

Jugular venipuncture to 8.5 ml blood tubes with ACD Solution A of trisodium citrate, 22.0g / L; citric acid, 8.0 g / L; and dextrose 24.5 g / L, 1.5mL as anticoagulant was used for blood samples collection.
RBC Lysis Solution (Qiagen, Hilden, Germany, #158902) was used to isolate peripheral blood lymphocytes. 1 ml of the buffy coat was transferred to 3 ml of RBC Lysis Solution in a conical tube, then incubated for either 5 or 20 minutes for adult horses or foals, respectively. In the next step, the tube was centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. Pellet of lymphocytes was washed with PBS three times, and during the last wash, cells were transferred to a 1.5 ml Eppendorf tube. Supernatant was removed, and the lymphocytes were snap-frozen in liquid nitrogen. Next, collected lymphocytes were transferred to the ultra freezer to—80°C and stored in these conditions until DNA isolation.
DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany, #69506) was used to isolate genomic DNA according to the manufacturer’s instructions. Obtained DNA was stored at -20°C until further analysis.

Thymus, spleen and inguinal lymph nodes were meshed through a 70 μm cell strainer (Corning) to obtain single-cell suspensions. For spleen, red blood cells (RBCs) were depleted using Qiagen RBC Lysis Solution according to manufacturer's instructions.

For detection of the transferred human MSCs in the peripheral blood of the recipient rat, one part of blood was collected in a tube containing EDTA immediately after sacrificed. Red blood cells were lysed by RBC Lysis Solution (Qiagen, Foster, CA), and cells were collected as a pellet after centrifugation. The cells were washed and resuspended in PBS, and then immunolabeled with purified mouse antihuman APC-conjugated CD44 or PE-conjugated CD105 monoclonal antibody (BD Pharmigen, San Diego, CA). Data were analyzed by flow cytometry following the manufacturer's instruction.
For detection of the transferred MSCs in the lung of the recipient rat, the lung tissue was fixed in 10% neutral buffered formalin and then embedded in paraffin. Thin sections of 5 µm thickness were obtained for immunohistochemical staining specific to human CD44 and CD105 (GeneTex, Irvine, CA), according to the manufacturer's protocol.

Genomic DNA was extracted from fresh whole blood (100-150 µl) of G1 male founders using RBC Lysis Solution (Qiagen, 158902) according to the instructions of the manufacturer. A NanoDrop spectrophotometer (Thermo Fisher) was used to determine gDNA concentration (total yield 2-20 µg gDNA). Whole-exome sequencing was performed as previously described (Wang et al., 2015 (link)) except that variants relative to the published NOD reference sequence (Steward et al., 2013 (link)) with quality scores ≥40 were annotated as potential mutations.