Hochest33342

BMDMs were pretreated with Tm (1 mg/ml) for 6 h prior to stimulation with LPS (100 ng/ml) 10 h. Cell apoptosis was measured by an Annexin V staining kit according to the manufacturer's instructions (eBioscience). Briefly, BMDMs in non-TC (tissue culture) treated dishes were detached by trypsinization and washed with PBS. Cells were resuspended in binding buffer and stained with Annexin-V-FITC solution. The proportion of Annexin V+ cells was calculated in the gated single cell population by BD LSRortessa flow cytometer with FlowJo software. The average fluorescence intensity of the Annexin V+ cells in each group was obtained in the gated Annexin V+ cell population and analyzed by FlowJo software. For PI immunofluorescence microscopy assay, the cells were washed with cold PBS and incubated with Hochest 33342 (10 μg/ml) and PI (1 mg/ml) solution (Solarbio) for 15 min. The cells were observed under Zeiss Vert.A1 microscope. Fluorescence images were captured with Zeiss Axiocam 503 color CCD camera controlled with ZEN software (ZEISS).

The transactivating transcriptional activator (TAT) peptide (YGRKKRRQRRRC) with purity ≥97% was purchased from ChinaPeptides Co., Ltd. (Shanghai, China). Taxol injection was obtained from Yangtze River Pharmaceutical (Group) Co., Ltd. (Jiangsu, China). Hochest33342 and 4% paraformaldehyde were provided by Solarbio Science and Technology Co., Ltd. (Beijing, China). Acetonitrile for high-performance liquid chromatography (HPLC) was obtained from Tedia Company, Inc (Fairfeld, OH, USA). Anhydrous pyridine, 1-(2-Hydroxyethyl)-1H-pyrrole-2,5-dione (HEMI), PEG (Mw2000) and D, L-lactide were obtained from Aladdin Bio-Chem Technology Co., LTD (Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM) (high glucose) cell culture medium, penicillin/streptomycin stock solutions, fetal bovine serum (FBS) and Trypsin were all bought from Gibco BRL (Gaithersberg, MD, USA). Protonic nuclear magnetic resonance (¹H-NMR) (400 MHz) was used to confirm the obtained compounds (Bruker AVVANCE DRX-400 NMR spectrometer, Bruker, Switzerland). In addition, gel permeation chromatography (GPC) (Waters 1515–2414, Waters, USA) was used to analyze all polymers by using tetrahydrofuran as the mobile phase (flow rate: 1 mL/min) at 30 °C. Polystyrene was used as standards.

The eversion of PS is one of the typical features of apoptosis. The apoptosis of cultured HT-22 was stained by FITC-labeled lactadherin (Haematologic Technologies, USA). Briefly, FITC-labeled lactadherin was added to the culture medium, incubated for 30 min at 37°C, and then washed to remove excess dyes. The nuclei were labeled with hochest 33342 (Solarbio, China). After being washed, Petri dishes were observed under an inverted fluorescence microscope.