Cd19 pc7 clone j3 119

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by density gradient centrifugation with Biocoll cell separation solution (Biochrom AG, Berlin, Germany) and frozen in FCS + 10% DMSO. For analysis, PBMCs were thawed and washed twice with PBS. One million cells were stained with smCD3-APC-H7 (clone SK7, BD), CD19-PC7 (clone J3-119, Beckman Coulter), CD20-V450 (clone 2H7, BioLegend), CD27-BV605 (clone L128, BD) and CD38-APC-R700 (clone HIT2, BD for 30 min at room temperature. Flow cytometry was performed on a BD FACSLyric^TM and data was analyzed using FlowJo software (version 10).

For each peripheral blood sample from CLL and HC-MBL patients, the CD5+ B cell population was purified on a FACSAria III (BD) flow cytometer. For this purpose, blood samples were labeled with CD45-V500-C (clone 2D1, BD); CD3-PB (clone UCHT1, BD); CD19-PC7 (clone J3-119, Beckman Coulter) and CD5-APC (clone L17F12, BD). The purity of the purified cell populations was always > 97%.

Detection of MAPK and NFκB activation was performed according to a previously published protocol⁴⁶ . Briefly, peripheral blood was stimulated with 10 μg/ml MDP (InvivoGen, San Diego, CA, USA) for 20 min at 37 °C or left unstimulated. Subsequently, the cells were fixed using 4% formaldehyde for 10 min at 25 °C, erythrocytes were lysed using 0.1% Triton X-100 (Sigma-Aldrich) for 15 min at 37 °C and the leukocytes were permeabilized using 80% ice-cold methanol for 30 min.
The following antibodies were used: CD3—A700 (clone MEM-57), CD14—PEDy594 (EXBIO) and CD19—PC7 (clone J3-119) (Beckman Coulter, USA, Brea, USA), phospho38 (Thr180)—A647 (#4552 S), phosphoErk1/2 (Thr202/Tyr204)—A488 (#4374 S), phosphoSAPJ/JNK (Thr183/185)—PE (#5755 S) (Cell Signaling, Denvers, MA, USA), phosphoNFκB—A647 (#4887) and anti-IκB—A488 (#5743) (both from Cell Signaling).