Immunophenotyping and Clonal Analysis of Multiple Myeloma

Primary MM cells were stained with Hoechst 33342 and fluorescent-labeled CD138 antibody and SP and NSP/CD138+ cells were isolated by cell sorting using FACS Aria. DNA isolation was performed using the QIAmp system (Qiagen, Valencia, CA, USA), the resultant DNA concentration was estimated by NanoDrop ND-1000 spectrophotometer, and DNA quality was assessed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). PCR primer sequences for IGH variable gene framework region 3 (FR3) analysis was designed as previously reported,^{23 (link)} and PCR was used to assess clonality using the BIOMED-2 system.^{24 (link)} PCR master mixes were purchased from Invivoscribe Technologies (San Diego, CA, USA), and PCR was performed as per the manufacturer’s instructions using HotStart Taq DNA polymerase (Qiagen).^{25 (link)}