The frequency of F-cells in transduced HSPCs undergoing erythroid differentiation was quantified as previously described.8 (link) Briefly, cells were fixed in 0.05% glutaraldehyde for 10 min, permeabilized with 0.1% Triton X-100 (Life Technologies) for 5 min, and stained with an anti-HbF APC antibody (Invitrogen) for 30 min. Cells were subsequently washed, acquired on an Accuri C6 flow cytometer (BD Biosciences), and analyzed using FlowJo software (v.10.8.1, BD Biosciences). To assess the impact of increased BACH2 expression on erythroid differentiation, transduced HSPCs undergoing erythroid differentiation were stained with a combination of anti-CD71 APC and CD235a PE (all from BD Biosciences) for 10 min. Stained cells were acquired on an Accuri C6 flow cytometer and analyzed using FlowJo software.
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Anti antibody Cd235a Cd71Cells Glutaraldehyde Triton x 100
Corresponding Organization : Harvard Stem Cell Institute
Other organizations :
Tohoku University, Siriraj Hospital, Mahidol University, University Medical Center Groningen, University of Groningen, Biogen (United States), British Heart Foundation, University of Cambridge, NHS Blood and Transplant, National Institute for Health Research, MRC Biostatistics Unit, Lund University, St. Jude Children's Research Hospital, University of Pittsburgh, University of Illinois at Chicago, University of Maryland, Baltimore, Duke Medical Center, Pacific Research Institute, Vitalant, University of California, San Francisco, UCSF Benioff Children's Hospital, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, Fundação Pró-Sangue Hemocentro de São Paulo, Fundação Centro de Hematologia e Hemoterapia de Minas Gerais, Hemorio, Kantonsspital Aarau, Aflac (United States), Emory University, Children's Healthcare of Atlanta, Health Data Research UK
Frequency of F-cells in transduced HSPCs undergoing erythroid differentiation
Erythroid differentiation of transduced HSPCs
control variables
Glutaraldehyde fixation (0.05% for 10 min)
Triton X-100 permeabilization (0.1% for 5 min)
Anti-HbF APC antibody staining (30 min)
Anti-CD71 APC and CD235a PE staining (10 min)
controls
No positive or negative controls were explicitly mentioned.
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