Streptavidin microbeads

CD4⁺ T cells were isolated from spleens using the MACS negative selection system (Miltenyi Biotec), by staining with biotinylated antibodies (Abs) against CD19 (6D5), CD8a (53–6.7), CD11b (M1/70), CD49b (DX5), I-A^b/d (25-9-17), and Ly-6G/Ly-6C (Gr-1, RB6-8C5) (BD Biosciences) followed by streptavidin-microbeads (Miltenyi Biotec). CD4⁺CD25⁻ cells were isolated by adding biotinylated anti-CD25 Ab (7D4) (BioLegend) to the first Abs. CD4⁺CD25⁺ cells were isolated using the MACS positive selection system by staining with PE anti-CD25 Ab followed by anti-PE microbeads (Miltenyi Biotec). Foxp3 was expressed in 96% of the CD4⁺CD25⁺ population. Purified CD4⁺ T cells, CD4⁺CD25⁻ Tc cells, or CD4⁺CD25⁺ Treg cells from WT mice were adoptively transferred into syngeneic nu/nu or TCRα^−/− mice by intraperitoneal injection (3.0 × 10⁶ cells/mice). In some experiments, donor T cells were labeled with CFSE, as previously described7 (link). Briefly, cells were incubated with 5 μM CFSE for 10 min at 37 °C. B cells were positively isolated using the MACS system by staining with biotinylated anti-CD19 Ab (1D3) (BD Biosciences) followed by streptavidin-microbeads.

Bone marrow cells were isolated from the tibia and femur and cultured in RPMI1640 medium with 10% FBS, 1% penicillin‐streptomycin, 55 µm β‐mercaptoethanol and 10% L929 conditioned media containing macrophage‐colony stimulating factor (M‐CSF) for 6 days to harvest BMDMs. Mouse T cells were isolated from spleens by using the Dynabeads Untouched Mouse T Cells Kit (Thermo Fisher). CD11b⁺Ly6C^hiLy6G^– cells were sorted on a MoFlo Astrios instrument. For CD11b⁺Ly6G⁺ isolation, cells were labeled with biotinylated anti‐Ly6G (BioLegend), incubated with streptavidin microbeads (BD), and separated on magnetic columns (Stemcell). For specific cell isolation from splenocytes, pDCs were isolated using anti‐mPDCA‐1 microbeads from Miltenyi Biotec (Auburn, CA). After pDCs isolation, macrophages were isolated with CD11b microbeads from Miltenyi Biotec, cDCs were isolated with mouse CD11c PE labeling and followed by PE selection cocktail from STEMCELL technologies, following the manufacturer's protocol. RNAs from pDCs, cDCs, and macrophages were isolated using TRIzol reagent (Invitrogen) and subjected to semi‐quantitative PCR analysis of 18S rRNA by using specific primer.

Cells were extracted from bone marrow (femur and tibia of both hind legs) of Hdac7^+/− and Hdac7^fl/− mice. Red blood cells were lysed with ACK lysis buffer. Cell counts were determined using a manual cell counter and Türk's staining to facilitate the counting of white cells nuclei. Isolated cells were incubated with anti-CD16/CD32 (2.4G2, Fc Block) (BD Bioscience) for 10 min on ice to reduce non-specific staining. The following antibodies were used for separation (from Miltenyi Biotec): anti-CD19-Microbeads (mouse), anti-Cd11b biotin (M1/70, BD Biosciences), and Streptavidin-Microbeads. Samples were incubated for 20 min at 4°C in the dark. CD11b needed double incubation, first with anti-Cd11b and second with Streptavidin-beads. Samples were put into Ls columns (Miltenyi Biotec) to perform magnetic cell separation. After three washes, cell from positive fractions (CD19+ and Cd11b+) were kept for further experimentation.