Primary hematopoietic stem cells were isolated from the bone marrow of female C57BL/6 mice (Jackson Labs; Ages 1–3 months) as described previously [34 (link)]. All animal experiments were conducted with permission obtained from the University of Illinois Institutional Animal Care and Use Committee (IACUC), Protocols #12–033, 14–1580. Lineage negative marrow was enriched using the EasySep Mouse Hematopoietic Progenitor Cell Enrichment kit (StemCell Technologies, Vancouver, Canada). HSCs were subsequently isolated via FACS as the Lin− Sca-1+ c-kit+ (LSK) sub-fraction using a cocktail of antibodies (eBioscience, San Diego, CA): PE-conjugated Sca-1 (1:100 dilution), APC-conjugated c-kit (1:100 dilution), and a 1:100 dilution of an FITC-conjugated Lineage (Lin) cocktail (CD5, B220, Mac-1, CD8a, Gr-1, Ter-119). LSK cells were sorted using a BD FACS Aria II flow cytometer (BD FACS Diva software).
Apc conjugated c kit
Manufactured by Thermo Fisher Scientific
The APC-conjugated c-kit is a fluorescently labeled antibody that binds to the c-kit receptor, also known as CD117. C-kit is a tyrosine kinase receptor expressed on the surface of various cell types, including hematopoietic stem cells, mast cells, and certain cancer cells. The APC (Allophycocyanin) fluorescent dye is conjugated to the c-kit antibody, enabling the detection and identification of c-kit positive cells using flow cytometry or other fluorescence-based techniques.
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Lab products found in correlation
Facsaria 2, by BD (2 mentions)
Easysep mouse hematopoietic progenitor cell enrichment kit, by STEMCELL (1 mentions)
Pe conjugated sca 1, by Thermo Fisher Scientific (1 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
Anti biotin microbeads for macs, by Miltenyi Biotec (1 mentions)
Pe cy7 conjugated sca 1, by BioLegend (1 mentions)
Facsdiva software, by BD (1 mentions)
Automacs pro separator, by Miltenyi Biotec (1 mentions)
2 protocols using apc conjugated c kit
1
Isolation of Murine Hematopoietic Stem Cells
2
Isolation and Purification of Hematopoietic Stem and Progenitor Cells
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For isolating HSPCs from bone marrow, magnetic‐activated cell sorting (MACS), and fluorescence‐activated cell‐sorting (FACS) analysis were performed as follow. Bone marrow cells were harvested from donor femur and tibia by crushing bones with mortar and pestle in PBS containing 2% FCS and filtering through a 40 μm mesh filter. After red blood cells were lysed, bone marrow cells were incubated with a cocktail of biotin‐conjugated lineage‐specific antibodies against CD45, Gr1, CD11b, Ter119, CD4, CD8, CD3 (553,029, 553,060, 553,086, 553,125, 553,309, 553,672, and 553,728, respectively, BD Biosciences), and anti‐Biotin microbeads for MACS (130–090‐485, Miltenyi Biotec). The separation of lineage‐negative cells was performed with an autoMACS pro separator (Miltenyi Biotec). Lineage‐depleted cells stained with APC‐conjugated c‐Kit (17–1171‐81, eBioscience) and PECy7‐conjugated Sca‐1 (108,116, Biolegend) antibodies were sorted using FACS Aria II (BD Biosciences) with over 95% purity. The DsRed fluorescence expression in the purified HSPCs was over 99% by FACS DiVa software (BD Biosciences).
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