Peripheral blood was harvested from healthy volunteers who donated to the University of Kansas Biospecimen Repository Core Facility (http://www.kumc.edu/school-of-medicine/biospecimen.html ). This facility is overseen by an inter-programmatic Internal Advisory Board (IAB) and the University of Kansas Medical Center Institutional Review Board (IRB). PBMCs were isolated by density gradient centrifugation using Histopaque (Sigma Aldrich). NK cells were then purified by negative selection using the Dynabeads Untouched Human NK cells kit (Invitrogen) following the manufacturer’s protocol. The purity of NK cells was assessed by flow cytometry to be >90% CD3−CD56+CD16+.
Dynabeads untouched human nk cells kit
Manufactured by Thermo Fisher Scientific
Sourced in France, United States
The Dynabeads™ Untouched™ Human NK Cells Kit is a magnetic bead-based system designed for the isolation of untouched natural killer (NK) cells from human peripheral blood mononuclear cells (PBMCs). The kit utilizes an antibody-coated magnetic bead technology to negatively select NK cells by removing non-target cells.
Automatically generated - may contain errors
Lab products found in correlation
Histopaque, by Merck Group (1 mentions)
Heparin vacutainers, by BD (1 mentions)
Lymphocyte separation medium, by Lonza (1 mentions)
Nucleospin rna xs, by Takara Bio (1 mentions)
Facsaria 2, by BD (1 mentions)
J 26s xp, by Beckman Coulter (1 mentions)
Lgm 3 medium, by Lonza (1 mentions)
Sanderson separation chamber, by Beckman Coulter (1 mentions)
Human ab serum, by Merck Group (1 mentions)
Penicillin streptomycin solution, by PAN Biotech (1 mentions)
Il 15, by R&D Systems (1 mentions)
Pancoll human, by PAN Biotech (1 mentions)
Interleukin 2 (il 2), by STEMCELL (1 mentions)
4 protocols using dynabeads untouched human nk cells kit
1
Isolation and Purification of Human NK Cells
2
Isolation of NK Cells from Breast Cancer Patients
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Peripheral blood of patients with breast cancer and healthy donors was directly obtained in heparin vacutainers, BD Biosciences, San Jose, CA, USA. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using lymphocyte separation medium, Lonza Walkersville, Inc. Houston TX, USA. The cell pellet was washed twice in calcium- and magnesium-free Dulbecco’s PBS-2% FCS-2 mM EDTA, enumerated on a haemocytometer with trypan blue and resuspended at 5 × 107/500 μl. NK cells were isolated through immunomagnetic negative selection using Dynabeads® Untouched™ Human NK Cells kit, Invitrogen Cergy-Pontoise, France, following the manufacturer’s recommendations. The purity (% of CD3−CD56+) of NK cells measured by flow cytometry confirmed that these cells were more than 98% CD3−CD56+ NK cells and less than 2% CD3+.
3
Enrichment and Sorting of Innate Lymphoid Cells
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Prior to FACS sorting, ILCs were enriched using Dynabeads™ Untouched™ Human NK Cells Kit (Invitrogen), allowing for the depletion of T, B, DCs, and macrophages. Enriched ILCs were labeled with flourochrome-conjugated monoclonal antibodies, listed in Supplementary Table 2 , and C10 (CD3−CD14−CD19−CD34−CD56BrightCD94+CD16−CD127− CD49a−), C2 (CD3−CD14−CD19−CD34−CD56BrightCD94+ CD16−CD127−CD49a+), and cNK (CD3−CD14−CD19− CD34− CD56DimCD94+CD16+), were sorted using the BD FACS Aria II (BD Biosciences) into fetal bovine serum for culture and activation or lysing buffer (NucleoSpin® RNA XS, Takara Bio USA Inc, Mountain View, CA) for RNA isolation.
4
Isolation and Culture of Human NK Cells
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Anticoagulated citrate dextrose-A-treated blood from healthy donors was purchased from the Regional Centre of Blood Donation and Blood Therapy in Krakow, Poland. Peripheral blood mononuclear cells (PBMC) were isolated by the standard density gradient centrifugation using Pancoll human (Panbiotech, Aidenbach, Germany). Lymphocytes were then separated from PBMC with the AVANTI J-26S XP elutriation system, equipped with the Sanderson separation chamber (Beckman Coulter, Brea, CA, USA), as described previously (24 (link)). NK cells were isolated from leukocytes using MACS technology and a Dynabeads™ Untouched™ Human NK Cells Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), in which NK cells were isolated via negative selection. NK cells were then cultured in LGM-3 medium (Lonza, Basel, Switzerland) supplemented with 5% v/v heat-inactivated human AB serum (Sigma, St. Louis, MO, USA), 500 U/ml IL-2 (STEMCELL Technologies), 140 U/ml IL-15 (R&D Systems, Minneapolis, MN, USA), and penicillin-streptomycin solution (100 U/ml and 100 µg/ml, respectively) (PAN Biotech GmbH, Bayern, Germany). Each experiment was performed using NK cells isolated from a different blood donor.
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