Annexin 5 and 7 aad staining kit

Three groups of 7-week-old female C57BL/6 mice were repetitively injected with PBS, 5 mg per kg of dNP2-dTomato or dNP2-ctCTLA-4, respectively, every other day for 14 days. The weight changes of the mice were daily monitored. At day 15, the mice were sacrificed and the morphologies of the spleen, liver and brain were carefully observed. The cytotoxicity of each protein against splenocytes and thymocytes was analysed using an Annexin V and 7-AAD staining kit (BD bioscience). The percentages of naive CD4 T cells in lymph nodes and spleen were analysed with isolated lymphocytes from each tissue after staining with 1/800 diluted anti-mouse CD4-PerCP-Cy5.5 (#45-0042), anti-mouse CD62L-FITC (#11-0621) and anti-mouse CD44-PE (#12-0441) FACS antibodies purchased from eBioscience. Liver toxicity of the proteins was analysed using an alanine aminotransferase activity assay kit (BioVision) and an aspartate aminotransferase activity assay kit (BioVision).

Cell proliferation was determined by MTS assay. MCF7 or MDA-MB-231 cells were seeded into 96-well plates in 100 μL of culture medium and cultured for the indicated time. Ten microliters per well of MTS (#G3582, Promega, Madison, WI) was added into each well for a 2-hour incubation at 37°C in a humidified, 5% CO₂ atmosphere. The absorbance was measured using a model ELX800 Micro Plate Reader (Bio-Tek Instruments, Winooski, VT) at 490 nm then the proliferation was calculated. Annexin V and 7-AAD staining kit (#559763, BD Biosciences, San Jose, CA) was used to detect apoptotic cells. WT, TRIM59 KD, or TRIM59 KO MCF7 cells were harvested with trypsin-EDTA and washed with PBS twice. Cells were then resuspended in 1× binding buffer at a concentration of 10⁶ cells/mL. A total of 5 μL Annexin V and 5 μL 7-AAD was added to 100 μL of the cell solution and incubated at RT for 15 minutes in the dark. Stained cells were washed and resuspended in 400 μL of 1× binding buffer and analyzed by flow cytometry.