Mouse pe positive selection kit

For HDAC3 qChIP, DP thymocytes were enriched using the EasySep Mouse Streptavidin RapidSpheres Isolation kit (#19860, StemCell Technologies) to remove SP and DN thymocytes with biotin-conjugated anti-CCR7 (4B12), anti-IL-7Rα (A7R34), anti-H2K (AF6-88.5), anti-CD44 (IM7), and anti-CD25 (PC61.5) as well as anti-TCRγδ (UC7-13D5) antibodies. For SP thymocyte enrichment, Mouse PE Positive Selection kit (#18554, StemCell Technologies) was used positively select for CCR7⁺ thymocytes (PE-conjugated anti-CCR7 (4B12)). ChIP was performed according to Pchelintsev et al. (2016) (link), with the following adjustments: After cell lysis and brief sonication (four mins, 30 s on/30 s off; Bioruptor Pico (Diagenode, Inc.)), equal volume of 2x MNase buffer (35 mM Tris-HCl pH 7.5, 25 mM NaCl, 120 mM KCl, 2 mM CaCl₂) was added to each sonication sample along with 3.75 U/μl of MNase (Cell Signaling Technologies #10011S) at 37°C for 15 mins. Anti-HDAC3 antibody (#85057, Cell Signaling Technologies) was used to isolate chromatin bound to HDAC3 after chromatin fragment size was confirmed to be less than 500 bp by gel electrophoresis. Isolated DNA was used to perform real-time PCR. Graphs depict fold enrichment over regions without HDAC3 binding (Rpl30 primers, Cell Signaling Technologies (#7015)). See Supplementary file 1 for primer sequences.

Hearts from WT mice 3 days post-MI were perfused with cold PBS, cut into small pieces, and dissociated with collagenase Ia (Sigma-Aldrich) at 37 °C for 30 min to obtain single-cell suspensions, which were then used for fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). For FACS, the cells were incubated with PerCP-Cy5.5-labeled anti-CD45 (550994, BD Biosciences, San Jose, CA, USA) and PE anti-F4/80 (12480182, eBioscience, San Diego, CA, USA) at 4 °C for 30 min in the dark, and the expression of the two molecules was analyzed on FACS Aria III (BD Bioscience). CD45^-, CD45⁺F4/80^-, and CD45⁺F4/80⁺ cell populations were collected, and CARD9 protein expression in these populations was assayed with western blotting. For MACS, the cells were first incubated with biotin anti-CD45 (13045182, eBioscience) and sorted using the Release Mouse Biotin Positive Selection Kit (STEMCELL, Seattle, WA, USA) to obtain CD45^- and CD45⁺ cells. Subsequently, CD45⁺ cells were incubated with PE anti- F4/80 and sorted using a mouse PE-positive Selection Kit (STEMCELL) to obtain CD45⁺F4/80^- and CD45⁺F4/80⁺ cells. Card9 mRNA level in these cells was evaluated by qPCR.