Anti human cd3 apc cy7 clone hit 3a

For immune complex formation 50 µg/ml ICAM1-Fc (produced in house, alternatively available from R&D Research, Wiesbaden, Germany) were mixed with 40 µg/ml APC-conjugated F(ab)₂ anti-human Fc antibody (Jackson Immunoresearch,) in assay buffer (Ca²⁺ and Mg²⁺-free PBS + 0.5% BSA) and incubated for 30 min at room temperature. AF488-labeled mAb24 anti-active LFA-1 antibody (BioLegend) was used at a 1:300 dilution. As LFA-1 conformation is temperature-dependent all cells were kept at 37 °C. Whole blood was pre-incubated or not with 20 ng/ml rhGDF-15 (Invigate, Lot 682039) for 10 min prior to adding an equal volume of stimulation and staining cocktail, thereby effectively diluting the GDF-15 concentration to a final of 10 ng/ml. CXCL12α was used at 200 ng/ml, αCD3/CD28 was diluted by 1:20. Anti-human CD3-APC/Cy7 clone HIT3a (BioLegend), anti-human CD4-BrilliantViolet711 clone A161A1 (BioLegend), anti-human CD8-PECy7 clone RPA-T8 (eBiosciene) defined the cells of interest. After staining, cells were fixed with 4% para-formaldehyde and stored overnight at 4 °C to promote erythrocyte lysis, before being analyzed on an Attune Nxt flow cytometer (ThermoFisher) employing a whole blood filter. To allow for statistical evaluation mean fluorescent intensity (MFI) values were normalized to control conditions by z-transformation. Samples were measured in duplicates.