Protocol detail

Quantification of Erythroid and Hematopoietic Stem Cells

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Spleen and bone marrow cells were collected and homogenized through a 70-µm filter. For the EB analysis, spleen cells were stained with FITC-conjugated anti-CD71 and PE-conjugated anti-TER-119 (BD Biosciences) after homogenization and washing (45 (link), 46 (link)). For the HSPC analysis, bone marrow cells were prepared for staining by erythrocyte lysis (BD Pharm Lyse; BD Biosciences). Cells were then stained in ice-cold PBS containing 2% FBS using the following antibodies: lineage cocktail containing TER-119, CD11b (Mac-1), Ly-6G/Ly-6C (Gr-1), CD3ϵ, and CD45R (B220); CD117 (Kit); Ly-6A/E (Sca-1); CD150 (Slamf1); CD48 (Slamf2); CD16/32; CD34; CD41 (Itga2b); and CD105 (endoglin) (BioLegend and eBiosciences). 4′,6 Diamidino-2-phenylindole (Invitrogen) was used for dead cell discrimination. SLAM and MKEP panels used were described previously (47 (link)–49 (link)). Samples were analyzed by flow cytometry using an LSR II (BD Biosciences). Post-acquisition analysis of data was performed with FlowJo software.

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Eaton N., Boyd E.K., Biswas R., Lee-Sundlov M.M., Dlugi T.A., Ramsey H.E., Zheng S., Burns R.T., Sola-Visner M.C., Hoffmeister K.M, & Falet H. (2022). Endocytosis of the thrombopoietin receptor Mpl regulates megakaryocyte and erythroid maturation in mice. Frontiers in Oncology, 12, 959806.

Publication 2022

FITC-conjugated anti-CD71 PE-conjugated anti-TER-119 BD Pharm Lyse 4′,6 Diamidino-2-phenylindole

Antibodies Bone marrow cells Cd11b Cd150 Cd71 Cell Cold Discrimination Endoglin Erythrocyte Fitc Flow cytometry Itga2b Mac 1 Sca 1 Slamf2 Spleen

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Variable analysis

independent variables

Spleen and bone marrow cell collection and homogenization

dependent variables

Erythroid blast (EB) analysis
Hematopoietic stem and progenitor cell (HSPC) analysis

control variables

70-µm filter for cell homogenization
Antibodies used for cell staining (FITC-conjugated anti-CD71, PE-conjugated anti-TER-119, lineage cocktail, CD117, Ly-6A/E, CD150, CD48, CD16/32, CD34, CD41, CD105)
Erythrocyte lysis for bone marrow cell preparation
4′,6 Diamidino-2-phenylindole for dead cell discrimination
Flow cytometry using an LSR II for sample analysis

positive controls

None specified

negative controls

None specified

Annotations

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