Coolcell lx

Mobilized peripheral blood and leukapheresis product were anonymously collected from donors undergoing stem cell mobilization at the Massachusetts General Hospital. Mononuclear cells were purified via Histopaque-1077 gradient (Sigma–Aldrich; 10771). CD34⁺ cells were isolated via positive magnetic bead selection using a CD34 MicroBead Kit (Miltenyi Biotec; 130-046-702) and LS columns (Miltenyi Biotec; 130-042-401), according to the manufacturer’s recommendations. CD34⁺ purity routinely exceeded 85%, as assessed by flow cytometry. Aliquots of 3–5 × 10⁵ cells were frozen in SFEM II medium (STEMCELL Technologies; 09655) + 10% DMSO (Sigma; 41640) + 20% fetal bovine serum (FBS; Gibco; 10082-147) using the CoolCell LX (Corning) at −80 °C, then transferred to liquid nitrogen cryogenic storage (VWR; CryoPro). Upon thawing, CD34⁺ cell viability was >90%, as assessed by trypan blue (Lonza; 17-942E).

Rag1^Cre::Rosa26^LSL-tdRFP mice were treated with 2 nmol calcipotriol (MC903, Tocris Bioscience) in 10 μL of 100% ethanol (EtOH) vehicle, or vehicle alone, on the bilateral ear skin daily for 7 days to induce AD-like inflammation. The next day, cervical sdLN were harvested and immediately homogenized manually through a 100 μm cell strainer (Fisher Scientific) into a 50 mL tube with the end of a plunger from a 3 mL syringe. The strainer was washed with wash medium (2% vol/vol FBS/PBS) and the strained cells were centrifuged at 400g for 5 minutes at 4°C. Next, cells were incubated with biotinylated antibodies (anti-mouse CD3e, CD19, CD11b; 1:300; Biolegend) in 100 μL of wash buffer for 20 minutes at 4°C, followed by two washes in 2 volumes of wash buffer. Next, no more than 10⁷ cells were incubated with Streptavidin MicroBeads (Miltenyi) in 500 μL separation buffer (0.5% w/v BSA in PBS; BSA and PBS from Sigma) at 4°C for 20 minutes, then added to LD columns (Miltenyi) pre-equilibrated with separation buffer and loaded in a QuadroMACS^™ Separator (Miltenyi) for negative cell selection. Remaining cells were eluted in 1 mL separation buffer and cells were centrifuged at 400g for 5 minutes at 4°C, followed by resuspension in freezing buffer (10% DMSO, Invitrogen; 20% FBS in DMEM, Sigma) and slow freezing to −80°C in a CoolCell^™ LX (Corning) device.

BM preparation was performed as described above. The resulting cell pellet was resuspended in RBC lysis buffer (buffer: 155 mM NH₄Cl + 10 mM KHCO₃ + 100 µM Na₂EDTA in H₂O; pH: 7.4) and centrifuged at 478× g for 10 min. After centrifugation, cells were resuspended in freezing medium (Recovery™ Cell Culture Freezing Medium, Thermo Fisher Scientific, Fremont, USA), transferred to cryovials, and placed in freezing containers (CoolCell™ LX, Corning, NY, USA) in a −80 °C freezer for at least 4 h before long-term storage in liquid nitrogen (−196 °C).
Samples were thawed by immersing the cryovials in a 37 °C water bath for 30 s. An amount of 100 µL of 10 mg/mL DNase I (Thermo Fisher Scientific, MA, USA) solution was immediately added to the cryovial, followed by 1000 µL of medium (DMEM + 10% FBS), both pre-cooled at 4 °C. The cell suspension was transferred to a tube containing 15 mL of pre-cooled medium (DMEM + 10% FBS) and centrifuged at 478× g for 10 min. The number of cells was determined using a Sysmex hemocytometer (XP-300™ Automated Hematology Analyzer, Sysmex Europe SE, Norderstedt, Germany). The cell pellet was resuspended in DMEM + 10% FBS. For cell separation using Parsortix, this suspension was transferred to a VacuTainer tube containing 1.8 mg/mL K2EDTA.