Pan t cell isolation kit 2

Spleens were dissociated with a syringe plunger, and single-cell suspensions were treated with ammonium-chloride potassium lysis buffer to remove erythrocytes.
Bone marrow cells were extracted from mice femur and tibia bones and were purified with CD3+ T isolated kit (CD3ϵ MicroBead Kit, mouse, 130-094-973, Miltenyi Biotec). Splenic CD4+ and CD8+ cells were purified in two steps: (1) Selection of CD4+ cells (CD4+ T Cell Isolation Kit, mouse, 130-104-454, Miltenyi) (2) Unbound cells were purified for CD8+ cells (CD8a+ T Cell Isolation Kit, mouse, 130-104-07, Miltenyi Biotec). For the tetramers binding reaction, we pooled splenocytes from previously vaccinated mice (5 mice after 8 days post infection) and purified their T cells using the untouched isolation kit (Pan T Cell Isolation Kit II, mouse, 130-095-130, Miltenyi Biotec).

Differentiation of murine T cells was performed as previously described (40 (link)). Splenic T cells were isolated by magnetic activated cell sorting using the “pan T cell isolation kit II” according to the manufacturer’s instructions (Miltenyi Biotech). Cells were fluorescently stained with an antibody cocktail containing αCD4-FITC (RM4-5, eBioscience), αCD44-PE (IM7, Biolegend), αCD62L-APC (MEL-14, eBioscience), and αCD25-PE-Cy5 (PC61.5, eBioscience) and were subsequently isolated by fluorescence activated cell sorting on MoFlo (Beckman-Coulter, Brea, CA, USA) in the FACS-core unit of the University Hospital Erlangen. Sorted naive T cells (CD4⁺CD62L⁺CD44^lowCD25⁻) were stimulated by plate-bound anti-CD3 (2 µg/ml, 145-2C11, BD Pharmingen, San Diego, CA, USA) and soluble anti-CD28 (2 µg/ml, 37.51, BD Pharmingen) and cultured for 4 days in the presence of IL-6 (40 ng/ml, R&D Systems, Minneapolis, MN, USA) and TGFβ1 (2 ng/ml, Biolegend) for Th17, IL-12p70 (20 ng/ml,R&D Systems) and anti-IL-4 (10 µg/ml, Biolegend) for Th1 or TGFβ1 (10 ng/ml, Biolegend) alone for Treg differentiation. T cell frequencies were analyzed by flow cytometry (FACSCantoII, BD Biosciences) using the antibodies listed in the section “Murine FACS analysis.”

For pDCs isolation, the spleens were dissociated into single-cell suspensions with spleen dissociation kit (130-095-926, Miltenyi Biotec, Bergisch Gladbach, Germany) on the gentleMACS Octo Dissociator with Heaters (130-096-427, Miltenyi Biotec, Bergisch Gladbach, Germany) running program 37C_m_SDK_1. For pan T cells isolation, the spleen single-cell suspensions were obtained by grinding followed by filtration through a nylon mesh. pDCs and pan T cells were purified from splenocytes using pDCs isolation kit (130-107-093, Miltenyi Biotec, Bergisch Gladbach, Germany) and pan T cell isolation kit II (130-095-130, Miltenyi Biotec, Bergisch Gladbach, Germany) respectively, according to the manufacturer’s instructions.

Primary T-cells were isolated from 3-4 mouse spleens by negative selection on magnetic bids using the Pan T cell Isolation Kit II (Miltenyi Biotech), see Grum-Schwensen et al. for details [13 (link)]. Purified T-cells were maintained in RPMI for 3 or 6 days with anti-CD3 or a combination of anti-CD3 and anti-CD28 antibodies, coupled to MACSibeads (Miltenyi Biotec) plus 10 ng/ml recombinant IL2 as described in [28 (link)].
Activated T-cells were stimulated with S100A4 protein (1 μg/ml) or S100A4 protein mixed with 6B12 antibody (6 μg/ml). Before fixation, PMA/Ionomycin and Golgistop™ (BD Biosciences) were added for 5 hours. Cells were washed with PBS and Fixable Viability Stain 450 (BD Biosciences) was added to discriminate between viable and dead cells.
Cells were fixed using the Cytofix/Cytoperm™ kit (BD Biosciences) and stained with the mouse Th1/Th2/Th17 phenotyping kit (BD Biosciences) containing antibodies against CD4, IL17A, IFNγ and IL4 according to the manufacturer’s instructions. Data acquisition and analysis were performed on a FACSVerse (BD Biosciences) using FlowJo software (Tree Star). All experiments were repeated 3-5 times.

Unstimulated mononuclear cells were collected via leukapheresis from a single healthy adult volunteer who provided written informed consent for the collection and use of these cells for research purposes under protocols approved by the Western Institutional Review Board (Olympia, WA). T-cells were enriched through magnetic cell sorting (Pan T-Cell Isolation Kit II; Miltenyi Biotec, Auburn, CA), and then frozen in aliquots and stored in liquid nitrogen. For use, cells were labeled with 3 μM CellVue Burgundy (eBioscience, San Diego, CA) [16 (link)].

Spleens from B6 donor mice were grounded and passed through a 40 µm cell strainer (BD Biosciences, San Jose, CA, USA) and resuspended in MACS buffer. Splenic T cell suspension was obtained using the Pan T cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). The kit is based on a cocktail of biotin-conjugated antibodies against CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, Anti-MHC-class II, and Ter-119. The isolation of T cells is achieved by depletion of magnetically labeled cells. T cell purity was evaluated by CD3⁺ staining and flow cytometry analysis and ranged from 72.9% to 74%. The number of injected T cells was adjusted according to acquired purity, ranging from 1.07 × 10⁶ to 1.06 × 10⁶ cells/100 µL injection volume.

MicroRNA and total RNA were extracted from WT and CD47⁻ T cell-derived EVs (isolated and purified as indicated in extended methods) using the miRNA easy kit from QIAGEN. The concentration and RNA quality were measured using a RNA-Bio analyzer. 100–200 ng of total RNA was used for first strand miRNA synthesis (Quanta Bioscience) as described (Kaur et al. 2016 (link)). First strand cDNA synthesis used a Maxima kit (2-Step RT PCR, Thermo Scientific) according to the manufacturer’s instructions or from Maxima first strand (Thermo scientific Fisher). U6 was used as a reference control gene for real -time PCR analysis as indicated in the legends. Cytoplasmic and nuclear RNA were extracted using the Cytoplasmic and Nuclear RNA Purification Kit from Norgen BioTek Corporation (Thorold ON). RNA was extracted according to manufacturer’s instructions. CD3⁺ cells from WT and Cd47 null mice (n = 3) were purified using Pan T Cell Isolation Kit II, mouse from Miltenyi Biotec, and mRNA isolation and real-time PCR were performed as described above using primers listed in Figure S7.