Anti mouse cd45 antibody

For cell-cell binding assays, RAW267.4 monocytes pre-labeled with Cell Tracker Red CMTPX Dye (Invitrogen, Carlsbad, CA) were co-cultured with confluent EpRAS cancer cells pre-treated with E-Cig 0.5% at a density of 2.5 × 10⁵ cells/cm² for 75 min to allow cell-cell binding. After the incubation, the co-culture was washed 3 times with 1X PBS to remove unbound monocytes, and fixed with 4% paraformaldehyde, followed by Hoechst 33342 staining. After three-washes, cells were visualized on fluorescent microscope. For inhibition study, RAW267.4 cells were pre-treated with 25 nM BIO 5192 (Tocris, Bristol, UK) for 4 h before co-cultured with EpRAS cells. For survival assays, the co-culture of non-labeled RAW267.4 monocytes and EpRAS cancer cells was further incubated with 100 ng/mL recombinant TRAIL (PeproTech Inc., Rocky Hill, NJ) to trigger apoptosis. After overnight treatment, the cells were washed 3 times with 1X PBS and harvested by Accutase solution. The single cell suspension was stained with anti-mouse CD45 antibody (BD Biosciences, San Jose, CA) and Annexin V apoptosis kit (Invitrogen, Carlsbad, CA) as per manufacturer’s instructions. The stained cells were analyzed with the LSR II flow cytometer (BD Biosciences, San Jose, CA). All experiments were independently repeated at least 2 times.

For single staining with the human CD45 antibody (Dako) or mouse lyve-1 (Abcam, San Fransisco, CA), the samples were incubated with antibody, then rinsed and a secondary reagent, AP-conjugated MACH2 Mouse Polymer (BioCare Medical, Concord, CA), RT for CD45 staining or EnVision + Dual Link- HRP Polymer (Dako) for lyve-1 staining was applied before development with Vulcan Fast Red Chromogen (BioCare Medical) or DAB+ (Dako). For dual staining of mouse and human CD45, anti-mouse CD45 antibody (BD Pharmingen, San Jose, CA) was applied, followed by a secondary rabbit anti-rat biotinylated antibody (Dako) and Streptavidin-HPR conjugated solution (Dako). The CD3, lyve-1 (Abcam), and CD19 (MyBiosource, San Diego, CA) antibodies were applied at the appropriate dilution, followed by HRP-conjugated EnVision + Dual Link System (Dako). In all cases, primary staining was developed using DAB+ chromogen solution (Dako).