Cd11a bv510

Multicolor flow cytometry analysis of BM was performed at the day of diagnosis to assess leukemic-associated immunophenotype (LAIP) of blasts cells. A following antibodies were used for cell immunophenotyping: CD34-PE, CD45-PerCP, CD10-PE-Cy7, CD19-APC, CD38-AlexaFluor-700, CD20-APC H7, CD11a-BV510 (BD Biosciences, Waltham, MA, USA), CD58 (Beckman Coulter, Brea, CA, USA). On the day 15th and 33rd BM was analyzed using the same panel of antibodies to determine MRD. To the appropriate amount of BM (10⁶ cells) antibodies listed above were added, samples were incubated for 20 min at room temperature in darkness. Erythrocytes were lysed for 10 min at room temperature in darkness with lysing solution (BD FACS Lysing Solution, Becton Dickinson Biosciences, San Jose, CA, USA), washed twice in PBS, and finally resuspended in 200 μL of PBS. For distinguishing nucleated cells, samples were stained with Syto^®41 (Thermo Fisher Scientific, Waltham, MA, USA). FACS analysis was done using FACSCanto or FACSCanto10 with FACSDiva Sorfware v. 8.1 (Becton Dickinson Biosciences, San Jose, CA, USA).

HO-1 expression was assessed in BM of 11 patients upon diagnosis. Then in patients in whom, after routine MRD staining, there was a sufficient material for additional staining, an intracellular HO-1 staining was performed (6 children at day 15, 3 children at day 33). In this purpose 10⁶ of bone marrow cells were stained with CD34-PE, CD45-PerCP, CD10-PE-Cy7, CD19-APC, CD38-AlexaFluor-700, CD20-APC H7, CD11a-BV510 (BD Biosciences, San Jose, CA, USA), lysed, fixed, and then permeabilized using a BD Intrasure Kit, according to the manufacturer’s instructions. After permeabilization step, cells were incubated with primary antibodies recognizing HO-1 (rabbit polyclonal, SPA 896, Enzo, Warszawa, Poland), washed twice, and stained with goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Life technologies, HITACHI, Tokyo, Japan). After two washing steps, samples were stained with Syto^®41 (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using FACSCanto10 with FACSDiva Sorfware v 8.0.1 (Becton Dickinson Biosciences, San Jose, CA, USA).