Anti human gitrl antibody

CRL5946 cells were seeded on eight-well culture slides (Corning Life Sciences) with each well 10⁴ cells and treated with cisplatin 2 ug/ml or γ-ray radiation 7.5 Gy. After incubating for 96 h, the medium was removed and the slide was washed twice with cold PBS. The viable cells were fixed with 4% paraformaldehyde for 10 min. The slides were stained with anti-human GITRL antibody (Clone:109101, R&D SYSTEMS), anti-mouse IgG Antibody-AF488 conjugated (Invitrogen), anti-human GITR antibody-PE conjugated (Clone#621, Biolegend Inc.), and DAPI (Cell Signaling) following the commercial instructions. The fluorescence images of whole sides were captured by Nikon A1R+ system, which is built onto a Nikon Eclipse TI-E inverted microscope with Nikon NIS Elements C for acquisition and analysis of images.

Paraformaldehyde was from Affymetrix (Santa Clara, CA, USA). Anti-OX40L antibodies were from Ancell Corporation (Bayport, MN, USA). Anti-human TACI, anti-human GITRL antibody and the respective isotype control were from R&D Systems (Minneapolis, MN). CD41a-PeCy5, and CD62P-FITC were from BD Pharmingen, CD3-APC/Fire, CD69-PE and CD25-FITC and CD56-PECy7 were obtained from BioLegend. The goat anti-mouse PE conjugate was from Dako (Glostrup, Denmark). Dead cells were excluded using Fixable Aqua (Invitrogen, Carlsbad, CA, USA) after extracellular staining according to the manufacturer’s instructions. Bicoll Separating Solution was purchased from Biochrom AG (Berlin, Germany).