Cd5 clone ucht2

NaYF₄: Er³⁺ and NaYF₄: Yb³⁺/Tm³⁺ nanoparticles were kindly provided by the electron microscopy room of Mudanjiang Medical University. 2-Morpholinoethanesulfonic acid (MES, 99%), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 98%) and N-hydroxysuccinimide (NHS, 98%) were purchased from Shanghai Aladdin Reagent Inc. (Shanghai, China). Hydrogen peroxide solution (H₂O₂, 30%) was purchased from Liaoning Quan Rui Reagent Co., Ltd. (Liaoning, China). CD20 (Clone: 2H7), CD20 (Clone: L26) and CD5 (Clone: UCHT2) monoclonal antibodies were purchased from BD Biosciences (San Jose, CA, USA). The Jeko-1 cell line was purchased from the Cell Resource Center of the Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai, China). The SP50B cell line was kindly provided by Prof. Tadashi Yoshino, Department of Pathology, Okayama University (Japan). SCID Beige SPF-grade mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China). RPMI 1640 culture medium and foetal bovine serum (FBS) were purchased from Gibco (USA).

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).