Peripheral blood samples were obtained from healthy donors and patients with AML at the City of Hope National Medical Center (COHNMC). Mononuclear cells were isolated using Ficoll separation. LSCs were sorted using a BD FACSAria™ Fusion (BD Biosciences). ILCs were isolated using EasySep™ Human Pan-ILC Enrichment Kit (STEMCELL) or were sorted using a BD FACSAria™ Fusion. Umbilical cord blood leukopaks were obtained from StemCyte, Inc (Baldwin Park, Californai). CD34+ cells were isolated using the CD34 MicroBead Kit (Miltenyi Biotec). All healthy donors and patients with AML signed an informed consent form. Sample acquisition was approved by the Institutional Review Board at the COHNMC.
Easysep human pan ilc enrichment kit
Manufactured by STEMCELL
The EasySep™ Human Pan-ILC Enrichment Kit is a laboratory tool designed to isolate and enrich innate lymphoid cells (ILCs) from human biological samples. It utilizes magnetic particles and a specialized buffer system to selectively deplete unwanted cell types, allowing for the isolation of the target ILC population.
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Lab products found in correlation
Cd34 microbead kit, by Miltenyi Biotec (1 mentions)
Facsaria fusion, by BD (1 mentions)
Ficoll histopaque 1077 gradient, by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Miltenyi Biotec (1 mentions)
Nk macs medium, by Miltenyi Biotec (1 mentions)
2 protocols using easysep human pan ilc enrichment kit
1
Isolation and Characterization of Leukemic Stem Cells
2
Isolation and Culture of Innate Lymphoid Cells
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Peripheral blood mononuclear cells (PBMC) were isolated on a Ficoll-Histopaque® 1077 gradient (Sigma-Aldrich, Lyon, France). From PMBCs derived from fresh blood, innate lymphoid cells were enriched by negative immunomagnetic selection (EasySep Human Pan-ILC Enrichment Kit, cat# 17975, StemCell). The kit contained a cocktail of magnetic antibodies targeting the major cell lines of the immune system except ILCs. Enrichment was performed according to the manufacturer’s instructions (36 ). Freshly enriched ILCs were cultured in a complete medium consisting of RPMI supplemented with 10% human serum AB (sHAB), 10 ng/mL IL-7 (PeproTech®, Neuilly sur Seine, France), 1 mM sodium pyruvate (Gibco) and antibiotics (penicillin 100 I.U./mL and streptomycin 100 μg/mL). Different combinations of media were tested in the presence of IL-2, IL-12, and/or IL-7 at different concentrations (at 5, 10, 20, 30, or 90 ng/mL) or a commercial medium specific for NK cells (NK MACS Medium, cat# 130-114-429, MiltenyiBiotec) with lower efficacy. From the ~60 donors, we obtained, after enrichment, between 2.7 x 105 and 9 x 105 cells/donor, with an average of 6.3 x 105 cells/donor. The percent yield of this enrichment protocol was from 0.02% to 0.7% with a mean of 0.18%. The predicted yield is 0.05 to 0.07% of mononuclear cells.
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