HDAC-IN-101

Guidelines (The following are our recommended protocols; this is a guideline and should be modified according to your specific needs).
1. Stock Solution Preparation
1.1 Solvent: For most dyes, organic solvents such as anhydrous DMSO are typically used for dissolution.
1.2 Concentration Recommendation: Generally, a high concentration of 1-10 mM stock solution is recommended.
1.3 Storage Conditions: Store at -20 ℃ or -80 ℃ protected from light and avoid repeated freeze-thaw cycles.
2. Working Solution Preparation
2.1 Diluent: Serum-free culture medium or PBS is typically used. Proteins and esterases in serum may interfere with staining or cause dye hydrolysis.
2.2 Working Concentration: 1-10 μM
Note: Please adjust the dye working solution concentration according to your actual situation and prepare fresh before use.
3. Cell Staining
3.1 Culture cells on sterile coverslips.
3.2 Remove the coverslips from the culture medium and aspirate excess medium.
3.3 Add working solution, gently shake to completely cover the cells, and incubate at 37 ℃ for 2-6 h.
3.4 Aspirate the dye working solution, wash 2-3 times with culture medium, 5 minutes each time, and observe using a fluorescence microscope or flow cytometry.
Note: If flow cytometry is required, the cells should be resuspended with trypsin before staining.

HDAC-IN-101 (Compound H6M) potently inhibits purified HDAC1 enzyme with an IC₅₀ of 65 nM^[1].

HDAC-IN-101 (72 h) inhibits the proliferation of HeLa, HepG2, HCT116, and H1299 cancer cells with IC₅₀ values of 3.59, 3.55, 3.23, and 2.57 μM, respectively, and shows lower cytotoxicity to normal LO2 cells with an IC₅₀ of 12.98 μM following 72 h of incubation^[1].

HDAC-IN-101 (5 μM) is pH independent, displaying minimal fluorescence intensity across pH 4.33 to 7.40 at a concentration of 5 μM when excited at 450 nm^[1].

HDAC-IN-101 (5 μM; 0-6 h) is activated by 10 μg/mL NTR and 500 μM NADH at pH 4.00, showing time-dependent fluorescence enhancement at 500 nm over 6 h of incubation^[1].

HDAC-IN-101 (H6M) shows high selectivity for NTR and low pH (4.00), exhibiting significant fluorescence enhancement at 500 nm only when treated with 10 μg/mL NTR and 500 μM NADH at pH 4.00^[1].

HDAC-IN-101 (1 μM; 0-4 h) selectively activates fluorescence in HeLa cells with high NTR expression, showing time-dependent enhancement over 4 h of incubation, while displaying minimal fluorescence in LO2 cells^[1].