Roboseptm buffer

One week after the third recall, lymphocytes were collected from spleen of immunized or control group mice. Pelleted cells were resuspended in RobosepTM buffer (Stemcell technologies) at a final concentration of 10⁸ cells/mL. 5*10⁸ cells per group were purified by CD3+, CD8+ or CD4+ negative selection using an EasySep isolation kit (Stemcell Technologies). Cells were then injected intravenously (IV) in 4–5 week old C57BL/6 mice (10⁷ cell in 300μl). The remaining cells were marked with α-CD3-FITC (1:1000, clone 145-2C11, eBioscience), α-CD4-BrilliantViolet421 (1:500, clone M1/70, Biolegend) or α-CD8-PerCP (1:500, clone 53–6.7, Biolegend) stain and analyzed by FACS to assess the purity of the cell suspension, purity was always superior to 95%.

A 37-micron reversible strainer was placed in each AggreWell™ 400 (Sigma, St. Louis, United States) well from which EBs were harvested, the EB suspension was transferred to a filter, and the well’s surface was rinsed with DMEM/F12 (Sigma, St. Louis, United States) to collect any remaining EBs. Flip the 37-micron mesh and place it on a clean centrifuge tube. Embryoid bodies collected on the filter were resuspended in a medium and incubated for 2 days. Half-chance the medium with 2.5 mL/well of EB Medium B every 2–3 days. Collagenase II solution was prepared (2,500 U/mL, #12,604-013; Thermo Fisher, United States). The EBs were resuspended in Collagenase II solution. Then, 3 mL of TrypLETMExpress (Thermo Fisher Scientific, United States) was added and mixed, and incubation was continued. After that, the cells were centrifuged at 200–300 g for 3–5 min. The cells were resuspended in RoboSepTM buffer (Stemcell, #20104) and incubated according to the EasySepTM human CD34 positive selection kit II (Stemcell, #17856) technical manual for CD34 positive cell sorting and purification.

Mice were sacrificed with a rising concentration of CO₂ and tibias, femur, iliac crests, and spine dissected and cleaned of remaining muscle and connective tissue. To extract Bone Marrow (BM) cells the cleaned bones were crushed in a pestle and mortar in a small volume of Iscove’s Modified Dulbecco’s Media (IMDM) and a single cell suspension generated by disaggregating with an 18 G needle and filtering through a 40 µm cell strainer (BD Biosciences, Wokingham, UK) into a centrifuge tube. The cells were centrifuged for 5 min at 1200 rpm and the pellet resuspended in 1 mL IMDM ready for use.
For immunomagnetic selection of HSPCs, BM cell numbers were determined using a Neubauer haemocytometer and cell densities were adjusted at a concentration of 1 × 10⁸ cells/mL in RoboSep^TM Buffer in a 14 mL polystyrene round-bottom tube (Thermo Fisher Scientific, Loughborough, UK) and 50 μL/mL of Normal Rat Serum added, and the tube placed in the RoboSep machine. Fully Automated Mouse hematopoietic progenitor cell isolation protocol #catalogno 19856 (Stem Cell Technologies, Cambridge, UK) was selected, the machine was loaded with antibody cocktail, RapidSpheres^TM, RoboSep^TM buffer and RoboSep^TM filter tips, following on-screen instructions. All reagents were obtained from Stem Cell Technologies, unless otherwise stated.