Facs arial 2

Single cell suspensions of thymocytes and splenocytes were obtained from 8 week old HDAC4 WT and HDAC4 KO mice. DN, DP, CD4⁺SP, CD8⁺SP T cells and NKT cells were sorted after anti-CD4, anti-CD8, anti-TCR-β and CD1d-tetramer antibody staining by BD FACSArialII (BD Biosciences). Sorted cells were collected with at least 95% purity.

For three-color immunolabeling, 1 × 10⁵cells were labeled with mAb mixture of peridinin chlorophyll protein (PeCy5)-conjugated anti-HLA-DR, APC-conjugated anti-CD11c and phycoerythrin (PE)-conjugated anti-CD123. At least 100,000cells were analyzed on a BD FACS Arial II. All of the above mAbs were bought from BD Biosource, USA. Analysis of flow cytometry data was ebioscience performed using Expo32 software.

The designed sgRNA sequence was inserted between two repeating coding sequences with 205 bp, and then, the compound sequence was inserted into the pT53 plasmid (Fig. 1C). When sgRNA plasmid and GFP reporter were co-transfected into 293 T cells, the sgRNA cleaves the sgRNA sequence of GFP reporter through homologous recombination of repeating sequence. Finally, a complete GFP sequence was generated, thus expressing GFP, proving the validity and activity of sgRNA designed. Briefly, to test the sgRNA activity, GFP reporter and sgRNA plasmids were co-transfected into 293 T cells by calcium phosphate precipitation. Briefly, 293 T cells were cultured in 12-well plates with 5 × 10⁵ cells per well on the day before transfection. 24 h after transfection, the medium was replaced with 750 µL fresh 293 T medium and 250 µL mixture that contained plasmids, CaCl₂, HEPES-buffered saline and ddH₂O. The plasmids included 1 µg GFP reporter and 1 μg sgRNA plasmids. The medium was replaced after 12 h, and a picture was taken under fluorescent microscopy (Olympus, X71, Japan) after 48 h. The cells were suspended in 800 µL of PBS for flow cytometry (BD FACS Arial II, US) analysis. The flow cytometry data were analyzed by C6 Channel.