HSCs expansion medium consists of BIT9500 (Stem Cell Technologies) supplemented with 50 ng/mL recombinant mouse (rm) SCF, 20 ng/mL FMS-like tyrosine kinase 3 ligand (Flt3L) and 10 ng/mL thrombopoietin (Tpo) (all from PeproTech, Inc.). p18SMI compounds were added where indicated. Bone marrow cells were harvested from C57BL/6 mice and made into a single-cell suspension. After immunomagnetic c-Kit enrichment (cKit-conjugated microbeads [MiltenyiBiotec, Bergisch-Gladbach]), cells were washed and resuspended in HSCs expansion medium. After culture, total nucleated cell counts were obtained, and a fraction of mononuclear cells (MNCs) or whole bone marrow cells were stained for flow cytometry analysis. Frequency of each cell population was determined by independently analyzing more than 5 × 105 cells per sample in triplicate.
Ckit conjugated microbeads
Manufactured by Miltenyi Biotec
CKit-conjugated microbeads are a type of magnetic beads designed for the isolation and enrichment of c-Kit (CD117) positive cells. They provide a simple and efficient tool for the separation and purification of cells expressing the c-Kit receptor, which is commonly found on various cell types, including hematopoietic stem and progenitor cells.
Automatically generated - may contain errors
2 protocols using ckit conjugated microbeads
1
Murine Hematopoietic Stem Cell Expansion
2
Isolation and Characterization of Neonatal Cardiac Cells
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The study was designed in accordance with and approved by the institutional review committees at San Diego State University along with the institutional review board (#120686) and no informed consent was required. Neonatal cells were derived from nonsurgically obtained postmortem cardiac tissue. Cells were isolated as previously published.12 Briefly, heart samples were mechanically minced into 1‐mm3 pieces and digested in collagenase II (150 U mg/mL, Worthington, LS004174) followed by brief low‐speed (850 rpm for 2 minutes) centrifugation to remove cardiomyocytes and tissue debris. The supernatant was subjected to magnetic‐activated cell sorting using c‐kit–conjugated microbeads (Miltenyi Biotec, #130‐091‐332), and c‐kit–enriched cells were plated in human c‐kit CIC media. The c‐kit–negative population was further purified by magnetic‐activated cell sorting for MSC surface markers CD90 and CD105. c‐kit CICs and MSCs were incubated at 37°C in 5% CO2 and used for cellular fusion between passages 5 and 10. Media used in the study are listed in Table S1 .
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