Cd19 clone hib19

PBMC of CLL patients and healthy donors were incubated with human IgG (Sigma-Aldrich, St. Louis, MO, USA) prior to the staining in order to minimize Fcɣ receptor binding, then washed and stained with the unconjugated GITR (clone 110416, R&D Systems, Inc., Minneapolis, MN, USA), GITRL (clone 109101, R&D Systems, Inc., Minneapolis, MN, USA), 4-1BB (clone 4B4-1, Ancell Corporation, Bayport, MN, USA), and 4-1BBL (clone C65-485, BD Pharmingen Inc., Heidelberg, Germany) mAbs or the isotype control at 10 μg/mL, followed by species-specific PE-conjugated detection antibodies (1:100). The CLL cells were then identified by staining for CD19 (clone HIB19, BD) and CD5 (clone UCHT2, BD), and dead cells were excluded based on 7-AAD (BioLegend, San Diego, CA, USA) positivity. FITC- and APC-conjugates (CD19 and CD5) were used in 1:25–1:50 dilutions, respectively. Specific fluorescence indices (SFIs) were calculated by dividing median fluorescence obtained with anti-GITR, anti-GITRL, anti-4-1BB, and anti-4 1BBL mAbs by median fluorescence obtained with the IgG1 isotype control. Positive expression was defined as SFI ≥1.5. Measurements were conducted using a BD FACSCanto™ II Flow Cytometer (BD Biosciences, Heidelberg, Germany) and data analysis was performed with FlowJo_V10.5.3 software (FlowJo LCC, BD).