Anti cd73 antibody

Isolated hAMC were seeded onto 24-well plates. After adherence, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. The fixative solution was removed and the cells were rinsed three times with PBS. The cells were processed with respective primary antibodies at 4°C overnight, including anti-CD105 antibody (mouse monoclonal, Invitrogen), anti-CD73 antibody (rabbit monoclonal, Abcam), and anti-vimentin antibody (mouse monoclonal, Santa Cruz). Then cells were incubated with corresponsive secondary antibodies conjugated to fluorescein (FITC, Jackson) or Alexa Fluor 488 (Jackson). Negative control was prepared by using isotype-controlled antibody. After washing three times with PBS, digital images were acquired with microscope.

Anti-mouse CD3 (clone 145-2C11) and anti-CD28 (clone PV-1) antibodies were prepared in our laboratory. DMEM, penicillin and streptomycin from GIBCO Inc. (Grand Island, NY, USA); fetal bovine serum (FBS) from HyClone Inc. (Logan, UT, USA); anti-mouse antibodies including Alexa 488 anti-Foxp3, -IFNγ and -perforin, PE anti-CD25, -CD39, -CD101, -CTLA-4, -granzyme B, ICOS and -IL-2, PerCP-Cy5.5 anti-CD73 and APC anti-CD45.2, FITC anti-human CD8, PE anti-human CD28, APC anti-human CD39 and PE-Cy7 anti-human CD73 from eBioscience (San Diego, CA, USA); PE anti-TNFα, -CCR6, -CD103, -Galectin-9 and -Helios antibodies from BioLegend (San Diego, CA, USA); PE anti-CD122, -CTLA-4, -LAG3 and -PD-1, APC anti-FR4, and PE-Cy7 anti-GITR antibodies from BD Bioscience (San Jose, CA, USA); Anti-CD73 antibody from Abcam (Cambridge, MA, USA); Mitomycin C, LPS, MTT, α, β-methylene adenosine 5’-diphosphate (AMPCP) from Sigma (St. Louis, MO, USA); KC7F2 from Calbiochem (Burlington, MA, USA); ZM241385 and MRS1754 from Tocris (Minneapolis, MN, USA); CFSE from Molecular Probes (Eugene, OR, USA); M2 peptides (LCMV gp33-41, KAVYNFATM) from AnaSpec, Inc. (San Jose, CA, USA); LY294002 from Cell signaling Technology (Danvers, MA, USA) were purchased.