NK cells were expanded as previously described [27 (link), 28 (link)]. Ficoll-purified PBMCs (20 ×106 cells) from healthy or ALL patients were co-cultured with 10×106 irradiated K562 clone 9. mblL21 artificial antigen-presenting cells (aAPC) for two weeks. K562 clone 9 cells were a kind gift from Dr. Dean A. Lee. Cells were grown in RPMI-1640 medium supplemented with 10% FBS (Sigma) and 1% penicillin/streptomycin (Hyclone). Fresh media was added every third day containing IL2 (100 U/mL; PeproTech). NK cells were purified at the end of the two-week coculture using MojoSort Human NK cell Isolation Kit (BioLegend). NK cells purity (>95%) was determined via flow cytometry (BD Accuri C6) using CD56 (5.11H11) and CD3 (HIT3a) antibodies from BioLegend.
Protocol detail
Expansion and Purification of NK Cells
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Antibodies
Antigen presenting cells
Cell isolation
Cells
Clone
Coculture
Ficoll
Flow cytometry
Human
K562 cells
Nk cells
Patients
Penicillin
Streptomycin
Corresponding Organization : Case Western Reserve University
Other organizations : Novartis (Switzerland), University Hospitals of Cleveland, Rainbow Babies & Children's Hospital
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Variable analysis
independent variables
- Healthy or ALL patient PBMCs
- Co-culture with irradiated K562 clone 9 mblL21 artificial antigen-presenting cells
- NK cell expansion
- RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin
- Addition of IL2 (100 U/mL) every third day
- NK cell purity (>95%) determined via flow cytometry using CD56 and CD3 antibodies
- Positive control: Healthy PBMCs co-cultured with irradiated K562 clone 9 mblL21 aAPC
- Negative control: Not explicitly mentioned
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