Mouse bone marrow was flushed from femurs and tibia to obtain bone marrow cells followed by isolation of CD117+ hematopoietic stem cells (HSCs) using CD117 beads (Miltenyi Biotec). The CD117+ HSCs were cultured in Stemspan medium (STEMCELL Technologies) in the presence of 10 μg/ml LDL, 10 ng/ml IL-3, 10 ng/ml IL-6, 40 ng/ml mSCF, 50 ng/ml TPO (STEMCELL Technologies) and 1% penicillin-streptomycin (VICMED) to induce HSCs differentiation. On day 3, megakaryocytes were collected using a discontinuous density BSA gradient method.
Cd117 beads
Manufactured by Miltenyi Biotec
CD117 beads are a laboratory tool designed for the specific isolation and enrichment of cells expressing the CD117 (also known as c-Kit) surface marker. These beads provide a reliable and efficient means to separate and purify CD117-positive cells from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Stemspan medium, by STEMCELL (1 mentions)
Interleukin 3 (il 3), by STEMCELL (1 mentions)
Interleukin 6 (il 6), by STEMCELL (1 mentions)
Hil 6, by Thermo Fisher Scientific (1 mentions)
Flt3l, by Thermo Fisher Scientific (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Stemspan media, by STEMCELL (1 mentions)
2 protocols using cd117 beads
1
Isolation and differentiation of mouse hematopoietic stem cells
2
CRISPR-mediated Gene Editing in HSCs
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CRISPR sgRNA sequences were designed using the Broad Institute sgRNA Designer. Forward and reverse oligonucleotide sequences including the guide sequence were synthesized, phosphorylated, annealed and individually cloned into lentiGuide-Cherry. Lentivirus was produced and B cells or HSCs were spin-infected as described above. For gene targeting in HSCs, the pMD2.G envelope was used, and for primary B cells, the pHIT123 envelope was used. HSCs were isolated using CD117 beads (Miltenyi Biotech) following manufacturer's instructions. Spin-infected HSCs were cultured overnight in StemSpan media (StemCell Technologies) in the presence of 100ng/ml mSCF, 6ng/ml IL-3, 10ng/ml hIL-6, 20ng/ml TPO, 60ng/ml Flt3L (all Peprotech).
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