Isolation and Purification of Rhesus Macaque HSPCs

Rhesus macaque PBMCs were isolated from PB using Ficoll-Paque PLUS density gradient medium (GE Healthcare) following manufacturer recommendations. G-CSF-mobilized (Amgen) and plerixafor-mobilized (Amgen) rhesus macaque CD34⁺ HSPCs were collected as described previously.²⁹ (link)^,⁶⁰ (link) In short, the animals were treated with a 5-day course of 15 μg/kg G-CSF (Amgen) subcutaneously and a single subcutaneous dose of 1 mg/kg AMD3100 (Sigma-Aldrich) on the morning of the fifth day, 3–4 h before leukapheresis. A small-volume leukapheresis procedure was performed using a CS3000 cell separator (Baxter Fenwal), and CD34⁺ HSPCs were immunoselected using a rhesus macaque CD34⁺ antibody (clone 12.8, Fred Hutchinson Cancer Research Center) and an anti-mouse IgM bead (Miltenyi Biotech). A CD34⁺ antibody (550761, BD Biosciences) was used to determine the purity of the immunoselected population.

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Peslak S.A., Demirci S., Chandra V., Ryu B., Bhardwaj S.K., Jiang J., Rupon J.W., Throm R.E., Uchida N., Leonard A., Essawi K., Bonifacino A.C., Krouse A.E., Linde N.S., Donahue R.E., Ferrara F., Wielgosz M., Abdulmalik O., Hamagami N., Germino-Watnick P., Le A., Chu R., Hinds M., Weiss M.J., Tong W., Tisdale J.F, & Blobel G.A. (2023). Forced enhancer-promoter rewiring to alter gene expression in animal models. Molecular Therapy. Nucleic Acids, 31, 452-465.

Publication 2023

Ficoll-Paque PLUS density gradient medium G-CSF AMD3100 CD34+ antibody

Amd3100 Animals Anti igm Antibody Cancer Cell Clone Ficoll G csf Leukapheresis Mouse Plerixafor Rhesus macaque

Corresponding Organization : National Institutes of Health

Other organizations : University of Pennsylvania, St. Jude Children's Research Hospital, Institute of Biophysics, Chinese Academy of Sciences, The University of Tokyo, Jazan University

Top 5 similar protocols

Variable analysis

independent variables

G-CSF mobilization
Plerixafor mobilization

dependent variables

Isolation of PBMCs from peripheral blood using Ficoll-Paque PLUS density gradient medium
Collection of G-CSF-mobilized and plerixafor-mobilized rhesus macaque CD34+ HSPCs

control variables

Rhesus macaque as the animal model
Manufacturer recommendations for Ficoll-Paque PLUS density gradient medium
Dose and duration of G-CSF and plerixafor treatment

controls

Positive control: G-CSF-mobilized rhesus macaque CD34+ HSPCs
Negative control: Not explicitly mentioned

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