Media199

CLL PBMCs were seeded at 5 × 10⁵ cells/200 μL of complete media (Media199, 10% fetal calf serum (FCS), penicillin/streptomycin and 5 ng/mL interleukin-4 [RayBiotech, Peachtree Corners, GA, USA]). Cells were cultured ±1 μM ODN2006 (TLR9 agonist; InvivoGen, San Diego, CA, USA) and incubated for 24 h. For FAK inhibition, CLL cells were pre-incubated for 2 h with Defactinib (Selleck Chemicals, Houston, TX, USA) at a range of concentrations (0.5 µM–5 µM). After 24 h, the amount of apoptosis was determined using a FITC-labeled Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend). The p-FAK levels in the CLL cells were measured by intracellular staining with a PE-conjugated anti-p-FAK antibody (BD Biosciences, Franklin Lakes, NJ, USA), after fixing and permeabilizing the cells using the True-Phos™ kit (BioLegend) according to the manufacturer’s instructions. The MFI values for p-FAK were determined in gated CD19+/CD5+ CLL cells using a Cytoflex LX flow cytometer (Beckman Coulter).

CLL PBMCs were seeded at 5 × 10⁵ cells/200 μL of complete media (Media199, 10% fetal calf serum (FCS), penicillin/streptomycin and 5 ng/mL interleukin-4 [RayBiotech]). Cells were cultured ±1 μM ODN2006 (TLR9 agonist; InvivoGen) ±1 μM defactinib (Selleck Chemicals) and incubated overnight. Transwell migration assays were performed using polycarbonate transwell inserts (5 μm pores) in 24-well plates (Corning Inc., Corning, NY, USA). A total of 600 μL complete media + 100 ng/mL CXCL12 (BioLegend) were added to the basolateral chambers, and PBMC (200 μL complete media) from CLL patients were transferred into the apical chambers and incubated for 4 h. Migrated CD19+/CD5+ CLL cells were quantified by volumetric counting using a Cytoflex LX flow cytometer (Beckman Coulter).