E obtained from German Cell Line collection (DSMZ, Braunschweig, Germany) Flow Cytometry Flow cytometry immunophenotyping of bone marrow (BM) aspirates was performed in at diagnosis and at relapse. Routine immunophenotypic classification using panel of monoclonal antibodies (moAbs) was performed as described previously [4]. Briefly, BM samples were stained with 2-, 3- and 4-color combinations of moAbs for 15 min in darkness, erythrocytes were lysed with NH4Clcontaining lysing solution for 15 min, washed and data were acquired using single FACS Calibur instrument throughout the study (BD Biosciences, San Jose, CA, USA) flow cytometer. Anti-CD66c (CEACAM6) moAb used in all diagnostic and relapse measurements in this study was clone KOR-SA3544 directly labeled PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28380356 to FITC (Immunotech, Marseille, France). Intracellular staining was performed using Fix Perm kit (Caltag, Burlingame, CA,Page 2 of(page number not for citation purposes)BMC Cancer 2005, 5:http://www.biomedcentral.com/1471-2407/5/pyrophosphate (Na4P2O7) and Complete Mini EDTA-Free (protease inhibitor cocktail tablets, Roche Diagnostics, Mannheim, Germany). Debris was sedimented by centrifugation for 3 min at 13000 rpm, 0 . Supernatants were mixed with 100 2?Laemmli’s SDS-polyacrylamide gel electrophoresis (PAGE) sample loading buffer, and heated for 5 min at 100 . Proteins were fractionated by SDS-PAGE on 12.5 gels and electrophoretically transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked for 1 h in PBS (pH 7.4) containing 0.5 Tween-20 and 5 nonfat dried milk. Blots were then incubated for 1 h at room temperature with antiKOR-SA3544 (Immunotech, Marseille, France) or antibeta-actin (Sigma-Aldrich, Saint Louis, MO, USA) moAbs and then developed using goat anti-mouse IgG (H+L)HRP conjugate (Bio-Rad). Immunoreactive material was then revealed by enhanced chemiluminescence (ECL, Amersham, Little Chalfont Buckinghamshire, UK) according to the manufacturer’s instructions.Isolation of RNA and Real-Time Quantitative PCR analysis (RQ-PCR) For RQ- PCR analysis, leukemic blasts were FACS sorted using sorting option on FACS Calibur or on FACS Aria instrument (1.1 ?104 – 4.7 ?105 cells from one patient). Isolation of RNA from FACS-sorted cells was performed using Trizol-reagent (Gibco BRL, Carlsbad, CA, USA) according to manufacturer’s instructions [17]. Complementary DNA was prepared using M-MLV Reverse Transcriptase (Gibco) according to manufacturer instructions. Glycogen (Gibco) 250 /mL was added when initial cell number was lower than 105. Quality of cDNA was verified by PCR on beta-2-microglobulin (B2M) HIV-1 integrase inhibitor 2 side effects housekeeping gene.Figure 1 CD66c positivity Correlation of ALL genotype categories and percentage of Correlation of ALL genotype categories and percentage of CD66c positivity. Median percentage of CD66cpos blasts is listed below each genotype group. Data of consecutive unselected patients with BCP ALL (n = 373) are shown.USA) according to manufacturer’s protocol. Acquired data was analyzed with Cell Quest (BD Biosciences) or Flow Jo (Tree Star, Ashland, OR, USA) software, lymphoblast gate was drawn based on optical scatter and CD19pos blast population was selected for further analysis. Value of 20 was chosen as a threshold of positivity as recommended by EGIL [16]. For robust prognostic significance testing, other threshold values were also tested as indicated in results.Cross-blocking of CD66c moAbs Bone marrow samples of CD66c positive blasts were st.
