Cytoflex 5 flow cytometer

A lyophilized vial of CFSE dye was reconstituted in 36 μL of DMSO to make a 5 mM solution. A 5 μM CFSE working solution was prepared in StemSpan SFEMII medium (09655, Stem Cell Technologies). Sorted LSK were resuspended in the later solution and incubated during 20 min at 37°C protected from light. The staining was quenched by adding 5 times the original staining volume of StemSpan SFEMII medium containing 10% FBS. Cells were incubated for additional 10 min, and for experiments were distributed in StemSpan SFEMII medium containing TPO (50 μl/mL), SCF (50 μl/mL), 1% L-glutamine (Gibco, 25030-081) and 2% Penicillin streptomycin (Gibco, 15140-122). Cells were maintained in culture at 5% CO₂ and 37°C for 7 days and collected. CFSE intensity was assessed in a CytoFlex 5 Flow Cytometer (Beckman Coulter). Data analysis was performed using FlowJo software version 10.5.3.

Cell cycle analysis in lymphoblast cell lines were thoroughly resuspended and fixed with ice-cold 70% ethanol and stored at −20 during 30 min. After fixation, cells were washed with PBS, stained with FxCycle PI/RNase staining solution (Invitrogen, F10797) and analyzed in a CytoFlex 5 Flow Cytometer (Beckman Coulter). Cell cycle analysis in mouse LT-HSC was performed using the cell surface markers PE-Cy7-Sca (Clone D7, 558162, BD Biosciences), APC-c-kit (Clone ACK2, 135108, BD Biosciences), FITC-CD150 (Clone A12 (7D4), 306306, Biolegend) and APC-Cy7-CD48 (Clone HM48-1, 470481-82, e-Bioscience), along with intracellular staining using PE-Cy7-Ki67 (350526, Biolegend) and DAPI (422801, Biolegend). Data analysis was performed using FlowJo software version 10.5.3.