Guava nexin reagent

Induction of apoptosis tests were carried out following the known procedure [42 (link)]. Apoptosis was determined by flow cytometric analysis of Annexin V and 7-aminoactinomycin D staining. After treatment cells during 24 h were harvested, washed 1–2 times with phosphate-buffered saline (PBS) and centrifuged at 400 g for 5 min. Cell pellets were resuspended in 200 μL of flow cytometry staining buffer (PBS without Ca²⁺and Mg²⁺, 2.5% FBS). Then, 200 μL of Guava Nexin reagent (Millipore, Bedford, MA, USA) was added to 5 × 10⁵ cells in 200 μL and the cells were incubated with the reagent for 20 min at room temperature in the dark. At the end of incubation, the cells were analyzed on NovoCyteTM2000 FlowCytometry System (ACEA).

Twenty thousand cells were seeded in 12-well plates and treated with 1 µM ZMC1 for 24 h. Cells were then trypsinized and resuspended in the original medium. Mix 100 µl of the resuspended cells with 100 µl of Guava Nexin Reagent (Millipore) in a 96-well plate and subject to Guava EasyCyte Flow Cytometer.

Initially, 5 × 10⁴ cells were seeded in a 6-well plate in 2 mL of full media. Mock-transfected and BCAT2 siRNA-transfected cells were washed twice with PBS, trypsinized, and collected by centrifugation. Afterwards, cells were incubated with Guava Nexin® Reagent (Millipore, USA) for 20 min at room temperature according to the manufacturer’s instructions. Samples were quantified by a Guava PCA flow cytometer (Millipore, USA). The data were analyzed by guavaSoft 2.7 Nexin software.

Cells were treated with BI 6727 (BT16 IC50=50nm; BT12 and MAF-A737 IC50= 24 nm), and then allowed to grow in normal culture medium for 24, 48 and 72 hours. The cell concentration was determined following staining with Guava ViaCount reagent (Millipore, Billerica, MA). Equal numbers of cells were then stained using Guava Nexin reagent (Millipore) to detect apoptotic cells. Samples were run on a Guava EasyCyte Plus flow cytometer (Millipore).

The apoptotic studies were carried out using Guava Nexin Reagent (Millipore). Cells with different types such as normal cells, early apoptotic cells and late apoptotic cells can be distinguished by this apoptosis study using flow cytometer60 (link). The Guava Nexin assay uses two stains (annexin V and 7-amino actinomycin D (7-AAD)) to quantitate the percentage of apoptotic cells. It was conducted according to the manufacturer’s protocol. In brief, after treatment with the alloys extracts for 24h, U2OS cells were collected and resuspended in 100ml medium supplemented with 100 μl 1% FBS. The cells were then incubated with 100 μl Annexin V-PE and 7-AAD labeling solution (Millipore) for 20 min at room temperature in the dark. Cells positive for AnV only (early apoptotic) or AnV and 7-AAD-positive cells (late apoptotic) were quantified by Guava EasyCyte 5HT flow cytometer. The data were analyzed by Guava Nexin Software v2.2.2.

Plasmid pET-28a was preserved in our laboratory. Taq DNA polymerase, DNA Ligation Kit, and restriction enzymes were obtained from Takara Biotech Co., Ltd. (Dalian, China). The Plasmid Mini Kit and Gel Extraction Kit were purchased from Omega (Norcross, USA). RPMI-1640 medium and DMEM/F12 (1:1) medium were from Hyclone (Logan, USA). Fetal bovine serum (FBS) was from Sangon biotech (Shanghai, China). Antibodies for His-tag and GRP78 were purchased from Proteintech (Wuhan, China). Antibody for GRP78 N-terminal was from Beyotime Biotechnology (Shanghai, China). Phycoerythrin-conjugated secondary antibody was obtained from Santa Cruz Biotechnology (Dallas, USA). Rhodamine Phalloidin was purchased from Cytoskeleton (Denver, USA). Guava Nexin Reagent and polyvinylidene difluoride (PVDF) membrane were from Millipore (Darmstadt, Germany). BCA protein assay kit and Immunol Staining Fix Solution were from Beyotime (Jiangsu, China). Enhanced chemiluminescence detection kit was from Engreen (Beijing, China). All other chemicals and reagents were obtained from Sigma (St. Louis, USA).