Donkey anti rabbit pe antibody

BM cells were flushed from the femurs of mice with PBS containing 2% fetal bovine serum (FBS), and the red blood cells were lysed with lysing buffer (eBioscience, San Diego, CA, USA). To examine the percentages of ST2⁺KSL cells, BM cells were suspended in PBS and incubated with PE-labeled anti-ST2, FITC-conjugated antibodies specific for lineage markers (CD3e, B220, TER119, CD11b, and Gr-1), PerCP-Cy5.5-conjugated anti-c-Kit, and APC-conjugated anti-Sca-1 antibodies (BioLegend) for 30 min. Cell apoptosis was measured using an annexin V apoptosis detection kit according to the instructions of the manufacturer (eBioscience). For analysis of phosphorylation AKT-S473, cells were fixed and permeabilized with the BD Cytofix/Cytoperm kit (BD Biosciences) and then stained with mouse anti-phospho-AKT-S473 PE or isotype control (eBioscience). For analysis of PUMA or p53 protein levels, cells were fixed in 4% paraformaldehyde and permeabilized in 0.25% saponin. Cells were stained with a primary anti-PUMA or anti-p53 antibody (MultiSciences Biotech Co., Ltd., Hangzhou, China) followed by a secondary donkey anti-rabbit PE antibody (BioLegend). Flow cytometric analysis was performed with FACSCalibur cytometer and CellQuest v3.3 software.

To examine the percentages of ST2⁺ cells, cells were suspended in PBS and incubated with PE-labeled anti-ST2 antibody (Biolegend) for 30 min. Cell apoptosis was measured using the Annexin V apoptosis detection kit (eBioscience) and in situ apoptosis detection kit (Roche) according to the manufacturer’s directions. For analysis of phosphorylation AKT-S473, cells were fixed and permeabilized with the BD Cytofix/Cytoperm kit (BD Biosciences) and then stained with mouse anti-phospho-AKT-S473 PE or isotype control (eBioscience). For analysis of RET, PUMA, or p53 protein levels, cells were fixed in 4% paraformaldehyde and permeabilized in 0.25% saponin. Cells were stained with a primary anti-RET, anti-PUMA or anti-p53 antibody (MultiSciences Biotech Co., Ltd, Hangzhou, China) followed by a secondary donkey anti-rabbit PE antibody (Biolegend). Flow cytometric analysis was performed with FACSCalibur cytometer (BD Biosciences) and CellQuest v3.3 software.